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Funder: NIHR Cambridge Biomedical Research centre, Background: Holoprosencephaly is a spectrum of developmental disorder of the embryonic forebrain in which there is failed or incomplete separation of the prosencephalon into two cerebral hemispheres. To date, dominant mutations in sonic hedgehog (SHH) pathway genes are the predominant Mendelian causes, and have marked interfamilial and intrafamilial phenotypical variabilities. Methods: We describe two families in which offspring had holoprosencephaly spectrum and homozygous predicted-deleterious variants in phospholipase C eta-1 (PLCH1). Immunocytochemistry was used to examine the expression pattern of PLCH1 in human embryos. We used SHH as a marker of developmental stage and of early embryonic anatomy. Results: In the first family, two siblings had congenital hydrocephalus, significant developmental delay and a monoventricle or fused thalami with a homozygous PLCH1 c.2065C>T, p.(Arg689*) variant. In the second family, two siblings had alobar holoprosencephaly and cyclopia with a homozygous PLCH1 c.4235delA, p.(Cys1079ValfsTer16) variant. All parents were healthy carriers, with no holoprosencephaly spectrum features. We found that the subcellular localisation of PLCH1 is cytoplasmic, but the p.(Cys1079ValfsTer16) variant was predominantly nuclear. Human embryo immunohistochemistry showed PLCH1 to be expressed in the notorcord, developing spinal cord (in a ventral to dorsal gradient), dorsal root ganglia, cerebellum and dermatomyosome, all tissues producing or responding to SHH. Furthermore, the embryonic subcellular localisation of PLCH1 was exclusively cytoplasmic, supporting protein mislocalisation contributing to the pathogenicity of the p.(Cys1079ValfsTer16) variant. Conclusion: Our data support the contention that PLCH1 has a role in prenatal mammalian neurodevelopment, and deleterious variants cause a clinically variable holoprosencephaly spectrum phenotype.

Inherited microcephaly exposes Zika culprit Microcephaly has been the terrifying hallmark of the recent outbreak of Zika virus (ZIKV) in the Americas. How the virus damages brain development in the fetus is enigmatic. Chavali et al. found that in congenital microcephaly, mutations in a neural precursor protein, Musashi-1 (MSI1), impede RNA binding to neural stem cell targets, resulting in abnormal brain development (see the Perspective by Griffin). MSI1 also binds ZIKV RNA to amplify viral replication in cells. This interaction could put a pregnant woman at risk of giving birth to a microcephalic child. Furthermore, MSI1 is expressed at high levels in the mouse testis, which may explain the sexual transmission of this virus. Science , this issue p. 83 ; see also p. 33

A five generation family with an X linked ocular disorder has been investigated. The major clinical features were reduced visual acuity, nystagmus, and myopia. Although impaired night vision was not a symptom, using psychophysical and electrophysiological testing both rod and cone function were found to be abnormal in all affected males. No abnormality was detected in carrier females. Gene location studies showed X linked transmission of a gene that maps to proximal Xp11. The findings observed in this cohort are similar to those previously reported in both congenital stationary night blindness type 2 (CSNB2) and Aland Island eye disease (AIED). This study addresses whether CSNB2 and AIED are a single entity or whether the latter is a subset of the former.

Autosomal recessive primary microcephaly (MCPH) is a disorder of neurodevelopment resulting in a small brain1,2. We identified WDR62 as the second most common cause of MCPH after finding homozygous missense and frame-shifting mutations in seven MCPH families. In human cell lines, we found that WDR62 is a spindle pole protein, as are ASPM and STIL, the MCPH7 and MCHP7 proteins3–5. Mutant WDR62 proteins failed to localize to the mitotic spindle pole. In human and mouse embryonic brain, we found that WDR62 expression was restricted to neural precursors undergoing mitosis. These data lend support to the hypothesis that the exquisite control of the cleavage furrow orientation in mammalian neural precursor cell mitosis, controlled in great part by the centrosomes and spindle poles, is critical both in causing MCPH when perturbed and, when modulated, generating the evolutionarily enlarged human brain6–9.

The division of the neocortex into functional areas (the cortical map) differs little between individuals, although brain lesions in development can lead to substantial re-organization of regional identity. We are studying how the cortical map is established in the human brain as a first step towards understanding this plasticity. Previous work on rodent development has identified certain transcription factors (e.g. Pax6, Emx2) expressed in gradients across the neocortex that appear to control regional expression of cell adhesion molecules and organization of area-specific thalamocortical afferent projections. Although mechanisms may be shared, the human neocortex is composed of different and more complex local area identities. Using Affymetrix gene chips of human foetal brain tissue from 8 to 12.5 post-conceptional weeks [PCW, equivalent to Carnegie stage (CS) 23, to Foetal stage (F) 4], human material obtained from the MRC-Wellcome Trust Human Developmental Biology Resource (http://www.hdbr.org), we have identified a number of genes that exhibit gradients along the anterior-posterior axis of the neocortex. Gene probe sets that were found to be upregulated posteriorally compared to anteriorally, included EMX2, COUPTFI and FGF receptor 3, and those upregulated anteriorally included cell adhesion molecules such as cadherins and protocadherins, as well as potential motor cortex markers and frontal markers (e.g. CNTNAP2, PCDH17, ROBO1, and CTIP2). Confirmation of graded expression for a subset of these genes was carried out using real-time PCR. Furthermore, we have established a dissociation cell culture model utilizing tissue dissected from anteriorally or posteriorally derived developing human neocortex that exhibits similar gradients of expression of these genes for at least 72 h in culture.

Duane's retraction syndrome (DRS) is a complex congenital eye movement disorder caused by aberrant innervation of the extraocular muscles by axons of brainstem motor neurons. Studying families with a variant form of the disorder (DURS2-DRS), we have identified causative heterozygous missense mutations in CHN1 , a gene on chromosome 2q31 that encodes α2-chimaerin, a Rac guanosine triphosphatase–activating protein (RacGAP) signaling protein previously implicated in the pathfinding of corticospinal axons in mice. We found that these are gain-of-function mutations that increase α2-chimaerin RacGAP activity in vitro. Several of the mutations appeared to enhance α2-chimaerin translocation to the cell membrane or enhance its ability to self-associate. Expression of mutant α2-chimaerin constructs in chick embryos resulted in failure of oculomotor axons to innervate their target extraocular muscles. We conclude that α2-chimaerin has a critical developmental function in ocular motor axon pathfinding.

The first haematopoietic stem cells in mammalian and non-mammalian vertebrates are derived from mesoderm, therefore genes that are important in mesoderm patterning and formation might also play an essential role in haematopoietic stem cell commitment and differentiation. Several members of the Wnt gene family are expressed in very specific patterns in embryonic mesoderm and have previously been shown to act as haematopoietic growth factors. In order to investigate in detail the role that such secreted proteins play in the biology of early haematopoietic commitment we have used in vitro differentiation of murine embryonal stem (ES) as a model system. Using reverse-transcriptase polymerase chain reaction analysis we identified several candidate Wnt genes whose expression pattern was consistent with a role in generation, maintenance and/or differentiation of early haematopoietic progenitor cells including three genes previously shown to have a role in haematopoiesis (Wnt5a, Wnt2b and Wnt10b). The most interesting candidate was Wnt3, because of its strong and regulated expression during in vitro differentiation of murine ES cells as well as its early embryonic expression in mesoderm. Overexpression of Wnt3 was sufficient to cause a consistent increase in the number of embryoid bodies committing to haematopoiesis further strengthening the evidence that this protein can enhance haematopoietic commitment during in vitro differentiation of ES cells. In addition, overexpression of Wnt3 caused a marked upregulation of Brachyury expression, thus providing some evidence that Brachyury may be one of the target genes for the Wnt3 signalling pathway.

Our current knowledge of mammalian forebrain development is meagre. The comparatively few relevant anatomical landmarks are, however, being supplemented by gene expression studies which are able to identify subsets of anatomical structures. We previously described cloning, subchromosomal localization and preliminary structural characterization of the human WNT8B gene, the first mammalian Wnt8b gene to be reported. Wnt genes encode intercellular signalling molecules which play a variety of critical roles in early development, including, in several cases, a presumed role in brain development. In the current report we present the full-length cDNA sequence and genomic organization of the human Wnt8b gene and report studies of expression of the Wnt8b gene in human and mouse embryos. The human and mouse expression patterns appeared identical and were restricted to the developing brain, with the great majority of expression being found in the developing forebrain. In the latter case expression was confined to the germinative neuroepithelium of three sharply delimited regions: the dorsomedial wall of the telencephalic ventricles (which includes the developing hippocampus), a discrete region of the dorsal thalamus and the mammillary and retromammillary regions of the posterior hypothalamus. Expression in the developing hippocampus may suggest a role for WNT8B in patterning of this region and subchromosomal localization of the human gene to 10q24 may suggest it as a candidate gene for partial epilepsy in families where the disease has been linked to markers in this region.

The RNA binding protein T-STAR was created following a gene triplication 520–610 million years ago, which also produced its two parologs Sam68 and SLM-1. Here we have created a T-STAR null mouse to identify the endogenous functions of this RNA binding protein. Mice null for T-STAR developed normally and were fertile, surprisingly, given the high expression of T-STAR in the testis and the brain, and the known infertility and pleiotropic defects of Sam68 null mice. Using a transcriptome-wide search for splicing targets in the adult brain, we identified T-STAR protein as a potent splicing repressor of the alternatively spliced segment 4 (AS4) exons from each of the Neurexin1-3 genes, and exon 23 of the Stxbp5l gene. T-STAR protein was most highly concentrated in forebrain-derived structures like the hippocampus, which also showed maximal Neurexin1-3 AS4 splicing repression. In the absence of endogenous T-STAR protein, Nrxn1-3 AS4 splicing repression dramatically decreased, despite physiological co-expression of Sam68. In transfected cells Neurexin3 AS4 alternative splicing was regulated by either T-STAR or Sam68 proteins. In contrast, Neurexin2 AS4 splicing was only regulated by T-STAR, through a UWAA-rich response element immediately downstream of the regulated exon conserved since the radiation of bony vertebrates. The AS4 exons in the Nrxn1 and Nrxn3 genes were also associated with distinct patterns of conserved UWAA repeats. Consistent with an ancient mechanism of splicing control, human T-STAR protein was able to repress splicing inclusion of the zebrafish Nrxn3 AS4 exon. Although Neurexin1-3 and Stxbp5l encode critical synaptic proteins, T-STAR null mice had no detectable spatial memory deficits, despite an almost complete absence of AS4 splicing repression in the hippocampus. Our work identifies T-STAR as an ancient and potent tissue-specific splicing regulator that uses a concentration-dependent mechanism to co-ordinately regulate regional splicing patterns of the Neurexin1-3 AS4 exons in the mouse brain., Author Summary Alternative splicing plays a key role in animal development and is largely controlled by the expression of RNA binding proteins. Most RNA binding proteins exist as families of sister proteins called paralogs, which result from gene amplification, including T-STAR, which is closely related to Sam68 and SLM-1. T-STAR, Sam68, and SLM-1 usually behave identically in splicing control in transfected cells. Here we report the physiological functions of T-STAR protein by knocking its parent gene out in the mouse. Surprisingly we observed no defects in germ cell maturation without T-STAR protein, an unexpected result given T-STAR protein is mainly expressed in the testis and its paralog Sam68 is essential for male fertility. Instead, we find T-STAR controls a panel of splicing targets that encode important synaptic proteins. T-STAR acts as a potent splicing repressor to establish regional splicing patterns of these target exons in the brain. Forebrain-derived structures like the hippocampus strongly express T-STAR protein to repress these target exons. Some T-STAR regulated splicing targets overlap with Sam68, but T-STAR also regulates its own distinct targets. Comparative genomic analyses are consistent with an ancient mechanism of splicing control by T-STAR that has been conserved since the radiation of bony vertebrates.

Coffin-Lowry syndrome (CLS; MIM 303600) is an uncommon X-linked disorder causing mental retardation and skeletal abnormalities. Most recently it was mapped to a 5.6-centimorgan (cM) region of Xp22, flanked distally by AFM291 wf5 and proximally by DXS1052 [Biancalana et al., 1994: Genomics 22:617–625]. We present information which supports this localization and further narrows the region to approximately 3.4 cM. A recombination in a carrier from a British family means that DXS365 is the closest proximal flanking marker identified to date for the region thought to contain the CLS gene. This information reduces the region of interest by approximately 2.2 cM, a significant decrease in terms of the scale of effort which will be required to isolate and analyze candidate genes. © 1995 Wiley-Liss, Inc.

Twenty-two X-linked cosmid clones have been localised by fluorescence in situ hybridisation (FISH). Twelve map to the long arm of the X chromosome and 10 to the short arm. Seven of the latter cosmids and an additional one were mapped by two colour FISH relative to reference markers DXS7 and DXS426 in proximal Xp. Finer localisation of one of the cosmids, namely HX43 was achieved by isolation of a microsatellite followed by genetic mapping with respect to reference markers in the region.

Joubert syndrome and related diseases (JSRD) are developmental cerebello-oculo-renal syndromes with phenotypes including cerebellar hypoplasia, retinal dystrophy and nephronophthisis (a cystic kidney disease). We have utilised the MRC-Wellcome Trust Human Developmental Biology Resource (HDBR), to perform in-situ hybridisation studies on embryonic tissues, revealing an early onset neuronal, retinal and renal expression pattern for AHI1. An almost identical pattern of expression is seen with CEP290 in human embryonic and fetal tissue. A novel finding is that both AHI1 and CEP290 demonstrate strong expression within the developing choroid plexus, a ciliated structure important for central nervous system development. To test if AHI1 and CEP290 may have co-evolved, we carried out a genomic survey of a large group of organisms across eukaryotic evolution. We found that, in animals, ahi1 and cep290 are almost always found together; however in other organisms either one may be found independent of the other. Finally, we tested in murine epithelial cells if Ahi1 was required for recruitment of Cep290 to the centrosome. We found no obvious differences in Cep290 localisation in the presence or absence of Ahi1, suggesting that, while Ahi1 and Cep290 may function together in the whole organism, they are not interdependent for localisation within a single cell. Taken together these data support a role for AHI1 and CEP290 in multiple organs throughout development and we suggest that this accounts for the wide phenotypic spectrum of AHI1 and CEP290 mutations in man.

Linkage analysis was carried out in two British families with incontinentia pigmenti (IP). Both showed exclusion at several markers in Xp and proximal Xq and showed probable linkage to the DXS52 and F8C loci in Xq28. This suggests that in these families the disease locus is IP2. Using a method based on the androgen receptor gene, and confirming the results where possible at the PGK-1 and DXS255 loci, it was shown that in affected females the maternally inherited X chromosome, where it could be identified, is inactive in the majority of cells.

We investigated three families whose offspring had extreme microcephaly at birth and profound mental retardation. Brain scans and postmortem data showed that affected individuals had brains less than 10\% of expected size (T (p.Ala29GlnfsX114) in a Turkish family and c.684\_685del (p.Pro229TrpfsX85) in two families of Pakistani origin. Using patient cells, we found that c.83+1G>T led to the use of a novel splice site and to a frameshift after NDE1 exon 2. Transfection of tagged NDE1 constructs showed that the c.684\_685del mutation resulted in a NDE1 that was unable to localize to the centrosome. By staining a patient-derived cell line that carried the c.83+1G>T mutation, we found that this endogeneously expressed mutated protein equally failed to localize to the centrosome. By examining human and mouse embryonic brains, we determined that NDE1 is highly expressed in neuroepithelial cells of the developing cerebral cortex, particularly at the centrosome. We show that NDE1 accumulates on the mitotic spindle of apical neural precursors in early neurogenesis. Thus, NDE1 deficiency causes both a severe failure of neurogenesis and a deficiency in cortical lamination. Our data further highlight the importance of the centrosome in multiple aspects of neurodevelopment.

Summary Genomic structural variants (SVs) are abundant in humans, differing from other variation classes in extent, origin, and functional impact. Despite progress in SV characterization, the nucleotide resolution architecture of most SVs remains unknown. We constructed a map of unbalanced SVs (i.e., copy number variants) based on whole genome DNA sequencing data from 185 human genomes, integrating evidence from complementary SV discovery approaches with extensive experimental validations. Our map encompassed 22,025 deletions and 6,000 additional SVs, including insertions and tandem duplications. Most SVs (53%) were mapped to nucleotide resolution, which facilitated analyzing their origin and functional impact. We examined numerous whole and partial gene deletions with a genotyping approach and observed a depletion of gene disruptions amongst high frequency deletions. Furthermore, we observed differences in the size spectra of SVs originating from distinct formation mechanisms, and constructed a map constructed a map of SV hotspots formed by common mechanisms. Our analytical framework and SV map serves as a resource for sequencing-based association studies.

none 49 We report that eight heterozygous missense mutations in TUBB3, encoding the neuron-specific beta-tubulin isotype III, result in a spectrum of human nervous system disorders that we now call the TUBB3 syndromes. Each mutation causes the ocular motility disorder CFEOM3, whereas some also result in intellectual and behavioral impairments, facial paralysis, and/or later-onset axonal sensorimotor polyneuropathy. Neuroimaging reveals a spectrum of abnormalities including hypoplasia of oculomotor nerves and dysgenesis of the corpus callosum, anterior commissure, and corticospinal tracts. A knock-in disease mouse model reveals axon guidance defects without evidence of cortical cell migration abnormalities. We show that the disease-associated mutations can impair tubulin heterodimer formation in vitro, although folded mutant heterodimers can still polymerize into microtubules. Modeling each mutation in yeast tubulin demonstrates that all alter dynamic instability whereas a subset disrupts the interaction of microtubules with kinesin motors. These findings demonstrate that normal TUBB3 is required for axon guidance and maintenance in mammals. none M. A. TISCHFIELD; H. N. BARIS; C. WU; G. RUDOLPH; L. VAN MALDERGEM; W. HE; WAI-MAN CHAN; C. ANDREWS; J. L. DEMER; R. L. ROBERTSON; D. A. MACKEY; J. B. RUDDLE; T. D. BIRD; I. GOTTLOB; C. PIEH; E. TRABOULSI; S. POMEROY; D. HUNTER; J. S. SOUL; A. NEWLIN; L. J. SABOL; E. J. DOHERTY; C. E. DE UZCATEGUI; N. DE UZCATEGUI; M. L. Z. COLLINS; E. C. SENER; B. WABBELS; H. HELLEBRAND; T. MEITINGER; T. DE BERARDINIS; A. MAGLI; C. SCHIAVI; M. PASTORE-TROSSELLO; F. KOC; A. M. WONG; A. L. LEVIN; M. T. GERAGHTY; M. DESCARTES; M. FLAHERTY; R. V. JAMIESON; H. U. MOELLER; I. MEUTHEN; D. F. CALLEN; J. KERWIN; S. LINDSAY; A. MEINDLI; M.L. GUPTA Jr; D. PELLMAN; E. C. ENGLE M. A. TISCHFIELD; H. N. BARIS; C. WU; G. RUDOLPH; L. VAN MALDERGEM; W. HE; WAI-MAN CHAN; C. ANDREWS; J. L. DEMER; R. L. ROBERTSON; D. A. MACKEY; J. B. RUDDLE; T. D. BIRD; I. GOTTLOB; C. PIEH; E. TRABOULSI; S. POMEROY; D. HUNTER; J. S. SOUL; A. NEWLIN; L. J. SABOL; E. J. DOHERTY; C. E. DE UZCATEGUI; N. DE UZCATEGUI; M. L. Z. COLLINS; E. C. SENER; B. WABBELS; H. HELLEBRAND; T. MEITINGER; T. DE BERARDINIS; A. MAGLI; C. SCHIAVI; M. PASTORE-TROSSELLO; F. KOC; A. M. WONG; A. L. LEVIN; M. T. GERAGHTY; M. DESCARTES; M. FLAHERTY; R. V. JAMIESON; H. U. MOELLER; I. MEUTHEN; D. F. CALLEN; J. KERWIN; S. LINDSAY; A. MEINDLI; M.L. GUPTA Jr; D. PELLMAN; E. C. ENGLE

Mutations in the gene encoding FERM domain-containing 7 protein (FRMD7) are recognized as an important cause of X-linked idiopathic infantile nystagmus (IIN). However, the precise role of FRMD7 and its involvement in the pathogenesis of IIN are not understood. In the present study, we have explored the role of FRMD7 in neuronal development. Using in situ hybridization and immunohistochemistry, we reveal that FRMD7 expression is spatially and temporally regulated in both the human and mouse brain during embryonic and fetal development. Furthermore, we show that FRMD7 expression is up-regulated upon retinoic acid (RA)-induced differentiation of mouse neuroblastoma NEURO2A cells, suggesting FRMD7 may play a role in this process. Indeed, we demonstrate, for the first time, that knockdown of FRMD7 during neuronal differentiation results in altered neurite development. Taken together, our data suggest that FRMD7 is involved in multiple aspects of neuronal development, and have direct importance to further understanding the pathogenesis of IIN.

Polymicrogyria (PMG) is a common malformation of the human cerebral cortex for which both acquired and genetic causes are known. Although genetic heterogeneity is documented, only one gene is currently known to cause isolated PMG. To clone new genes involved in this type of cerebral malformation, we studied a fetus presenting a defect of cortical organization consisting of a polymicrogyric cortex and neuronal heterotopia within the white matter. Karyotype analysis revealed that the fetus was carrier of a balanced, de novo, chromosomal translocation t(2;7)(q35;p22). Cloning and sequencing of the two translocation breakpoints reveals that the chromosomal rearrangement disrupts the coding region of a single gene, called NHEJ1, Cernunnos, or XLF, in 2q35. The NHEJ1 gene was recently identified as being responsible for autosomal recessive immunodeficiency with microcephaly. Using quantitative PCR experiments, we show that a truncated transcript is expressed in the polymicrogyric patient cells, suggesting a potential dominant negative effect possibly leading to a different phenotype. We performed in situ hybridization on human embryos and showed that the NHEJ1 transcript is preferentially expressed in the telencephalic ventricular and subventricular zones, consistent with the phenotype of the affected individual. In the human adult central nervous system (CNS), NHEJ1 is mainly expressed in the cerebral cortex and in the cerebellum. The association of PMG with the disruption of its transcript suggests that, in addition to its recently uncovered function in the immune system, the NHEJ1 protein may also play a role during development of the human cerebral cortex. Hum Mutat 28(4), 356–364, 2007. © 2006 Wiley-Liss, Inc.

A striking finding of the human and mouse genome sequencing projects is that, although there are many differences between the two species, they have similar numbers of genes. The differences arise during development and are driven, in part, by changes in gene expression. The MRC-Wellcome Trust Human Developmental Biology Resource (HDBR) is a unique resource that provides human embryonic and foetal tissues to the scientific community, enabling gene-expression studies at these crucial periods of development.

Wnt genes encode intercellular signalling molecules that play important roles in key processes of embryonic development such as mesoderm induction, specification of the embryonic axis, and patterning of the central nervous system, spinal cord, and limb. Multiple such genes are known to exist in each of several species that have been investigated, and they have been classified into various groups and subgroups on the basis of high sequence homology and common expression patterns. The vertebrate Wnt8 subfamily includes genes from Xenopus, zebrafish, and chicken, but, to date, no mammalian homologues have been described. We now report cloning and characterization of a novel human member of this family that we have termed WNT8B on the basis of the very high sequence similarity of the inferred protein to those encoded by the Xenopus and zebrafish Wnt8b genes. PCR typing of a human monochromosomal hybrid cell panel mapped the gene to chromosome 10, and FISH mapping provided a subchromosomal location at 10q24. Northern blotting and RT-PCR assays indicated that the WNT8B gene is expressed in several human tissues during fetal and adult stages.

Cornelia de Lange syndrome (CdLS) is a rare developmental malformation syndrome characterised by mental handicap, growth retardation, distinctive facial features and limb reduction defects. The vast majority of CdLS cases are sporadic. We carried out a high density bacterial artificial chromosome (BAC) microarray comparative genome hybridisation screen but no evidence was found for a consistent pattern of microdeletion/micro-duplication. As an alternative, we focused on identifying chromosomal regions spanning associated translocation breakpoints. We prioritised the distal 3q region because of the occurrence, in a classical CdLS patient, of a de novo balanced translocation with a breakpoint at 3q26.3 and of reports of phenotypic overlap between cases of mild CdLS and individuals trisomic for the 3q26-q27 region. We show that the 3q26.3 breakpoint severs a previously uncharacterised giant gene, NAALADL2, containing at least 32 exons spanning 1.37 Mb. Northern blot analysis identified up to six different transcripts in the 1–10 kb range with strongest expression in kidney and placenta; embryonic expression was largely confined to duodenal and stomach endoderm, mesonephros, metanephros and pancreas. Transcript analysis identified extensive alternative splicing leading to multiple 5′ and 3′ untranslated regions and variable coding sequences. Multiple protein isoforms were defined by different N-terminal regions (with at least four alternative initiating methionine codons), and by differential protein truncation/use of alternative C-terminal sequences attributable to alternative splicing/polyadenylation. Outside the N-terminal regions, the predicted proteins showed significant homology to N-acetylated alpha-linked acidic dipeptidase and transferrin receptors. Mutation screening of NAALADL2 in a panel of CdLS patient DNA samples failed to identify patient-specific mutations. We discuss the possibility that the 3q26.3 translocation could nevertheless contribute to pathogenesis.

Five polymorphic CA-dinucleotide repeats, identified in cosmids from the short arm of the human X chromosome, have been characterized and localized to Xp21.1 (DXS572), Xp11.4 (DXS556, DXS574), and Xp11.22–p11.23 (DXS722, DXS573). Genetic mapping with respect to five reference markers that include the gene for CGD (CYBB in Xp21.1), complemented by physical mapping information, has indicated the order tel - DXS572 - CYBB - DXS1110 - DXS556 - DXS574 - DXS7-DXS426-DXS722-DXS573-DXS255-cen.

Autosomal dominant sacral agenesis is characterised by a partial agenesis of the sacrum typically involving sacral vertebrae S2-S5 only. Associated features include anorectal malformation, a presacral mass, and urogenital malformation. Together, these features have been defined as the Currarino syndrome. Recently, HLXB9 has been identified as the major causative gene in Currarino syndrome allowing identification of asymptomatic heterozygotes. In this review, we have performed an analysis of medical publications, and our own additional cases, to identify the range of malformations and complications that occur. We have also estimated risks of malformation in heterozygotes by using Weinburg's proband method on families personally known to us in order to provide accurate genetic counselling information. Keywords: sacral agenesis; presacral mass; anorectal malformation; Currarino triad

Turner syndrome is characterized by short stature and is frequently associated with a variable spectrum of somatic features including ovarian failure, heart and renal abnormalities, micrognathia, cubitus valgus, high-arched palate, short metacarpals and Madelung deformity. Madelung deformity is also a key feature of Leri-Weill syndrome. Defects of the pseudoautosomal homeobox gene SHOX were previously shown to lead to short stature and Leri-Weill syndrome, and haploinsufficiency of SHOX was implicated to cause the short stature phenotype in Turner syndrome. Despite exhaustive searches, no direct murine orthologue of SHOX is evident. SHOX is, however, closely related to the SHOX2 homeobox gene on 3q, which has a murine counterpart, Og12x. We analysed SHOX and SHOX2 expression during human embryonic development, and referenced the expression patterns against those of Og12x. The SHOX expression pattern in the limb and first and second pharyngeal arches not only explains SHOX -related short stature phenotypes, but also for the first time provides evidence for the involvement of this gene in the development of additional Turner stigmata. This is strongly supported by the presence of Turner-characteristic dysmorphic skeletal features in patients with SHOX nonsense mutations.

Our understanding of early human development has been impeded by the general difficulty in obtaining suit-able samples for study. As a result, and because of the extraordinarily high degree of evolutionary conserva-tion of many developmentally important genes and developmental pathways, great reliance has been placedon extrapolation from animal models of development, principally the mouse. However, the strong evolution-ary conservation of coding sequence for developmentally important genes does not necessarily mean thattheir expression patterns are as highly conserved. The very recent availability of human embryonic samplesfor gene expression studies has now permitted for the first time an assessment of the degree to which we canconfidently extrapolate from studies of rodent gene expression patterns. We have found significant human–mouse differences in embryonic expression patterns for a variety of genes. We present detailed data for twoillustrative examples. Wnt7a, a very highly conserved gene known to be important in early development,shows significant differences in spatial and temporal expression patterns in the developing brain (midbrain,telencephalon) of man and mice. CAPN3, the locus for LGMD2A limb girdle muscular dystrophy, and itsmouse orthologue differ extensively in expression in embryonic heart, lens and smooth muscle. Our studyalso shows how molecular analyses, while providing explanations for the observed differences, can be impor-tant in providing insights into mammalian evolution.INTRODUCTIONAlthough the structural genomics phase of the human genomeproject is nearing completion, and will shortly provide a completeinventory of human genes, our knowledge of human gene expres-sion is rudimentary (1). High resolution gene expression studiesare most conveniently carried out on the miniaturized tissues ofthe embryo, but the general difficulty in accessing suitable mate-rial for studying early human development and the widespreadbelief that early mammalian development is essentially conservedhave prompted widespread studies of rodent models, notably themouse. The resulting reliance on rodent embryonic expressionstudies makes it important to know just how far we can extrapo-late from them with confidence.The huge emphasis on gene expression in rodent embryos hasbeen justified by strong similarities in the organization andmorphology of vertebrate embryos and by the extraordinaryevolutionary conservation of many developmental pathways andof key genes that are known or expected to be important in regu-lating early development. Species differences in development canbe represented by an hourglass model (2,3): very considerabledifferences exist at very early stages in development, such asgastrulation and neurulation, and morphological divergence canbe extensive in later development,but at intermediate stages whenthe body plan is being laid down embryonic development hasbeen widely considered to be highly conserved during vertebrateevolution. However, the idea that there is a conserved embryonic(phylotypic) stage in vertebrates has not been supported bydetailed comparative studies: stage-matched embryos fromclosely related vertebrate and mammalian species were recentlyshown to exhibit considerable morphological differences (4).Recent molecular studies have also emphasized unexpectedspecies differences and raised concerns regarding the extent to

The HLXB9 homeobox gene was recently identified as a locus for autosomal dominant Currarino syndrome, also known as hereditary sacral agenesis (HSA). This gene specifies a 403–amino acid protein containing a homeodomain preceded by a very highly conserved 82–amino acid domain of unknown function; the remainder of the protein is not well conserved. Here we report an extensive mutation survey that has identified mutations in the HLXB9 gene in 20 of 21 patients tested with familial Currarino syndrome. Mutations were also detected in two of seven sporadic Currarino syndrome patients; the remainder could be explained by undetected mosaicism for an HLXB9 mutation or by genetic heterogeneity in the sporadic patients. Of the mutations identified in the 22 index patients, 19 were intragenic and included 11 mutations that could lead to the introduction of a premature termination codon. The other eight mutations were missense mutations that were significantly clustered in the homeodomain, resulting, in each patient, in nonconservative substitution of a highly conserved amino acid. All of the intragenic mutations were associated with comparable phenotypes. The only genotype-phenotype correlation appeared to be the occurrence of developmental delay in the case of three patients with microdeletions. HLXB9 expression was analyzed during early human development in a period spanning Carnegie stages 12–21. Signal was detected in the basal plate of the spinal cord and hindbrain and in the pharynx, esophagus, stomach, and pancreas. Significant spatial and temporal expression differences were evident when compared with expression of the mouse Hlxb9 gene, which may partly explain the significant human-mouse differences in mutant phenotype.

Partial absence of the sacrum is a rare congenital defect which also occurs as an autosomal dominant trait; association with anterior meningocoele, presacral teratoma and anorectal abnormalities constitutes the Currarino triad (MIM 176450). Malformation at the caudal end of the developing notochord at approximately Carnegie stage 7 (16 post-ovulatory days), which results in aberrant secondary neurulation, can explain the observed pattern of anomalies. We previously reported linkage to 7q36 markers in two dominantly inherited sacral agenesis families. We now present data refining the initial subchromosomal localization in several additional hereditary sacral agenesis (HSA) families. We excluded several candidate genes before identifying patient-specific mutations in a homeobox gene, HLXB9, which was previously reported to map to 1q41-q42.1 and to be expressed in lymphoid and pancreatic tissues.

The Wnt gene family encodes a set of signalling molecules, thought to play an important role in key processes of embryonic development. In vertebrates as a whole 20 different Wnt genes have been identified to date, however, a complement of only 16 have been identified in man and for some of these the complete coding sequences are unavailable. We have recently isolated the full-length cDNA sequence of a new human WNT gene, WNT11, investigated its genomic organisation and performed detailed expression studies in early human embryos. These have shown that the expression of human WNT11 is restricted to the perichondrium of the developing skeleton, lung mesenchyme, the tips of the ureteric buds and other areas of the urogenital system and the cortex of the adrenal gland. This, for the first time, provides information for the embryonic expression of human WNT11. We have mapped WNT11 to 11q13.5 and this together with its expression in the perichondrium of the developing skeleton, makes it a plausible candidate gene for HBM, which has been previously linked to markers from this region.

AIMS OF THE STUDY: To identify the proportion and type of deletions present in the glypican 3 (GPC3) gene in a group of patients with Simpson-Golabi-Behmel syndrome (SGBS). SUBJECTS AND METHODS: PCR analysis using primer pairs which amplify fragments from each of the eight exons of the GPC3 gene was carried out in a series of 18 families with SGBS (approximately half of reported cases). RESULTS: Deletions were detected in only five families (one reported previously). We found deletions in all exons of the gene except exon 3. CONCLUSIONS: Our results suggest that large scale deletions may be less common in SGBS than was originally thought. One patient, with an exon 4 and 5 deletion, lacked the characteristic facial dysmorphic features. This raises the possibility of involvement of GPC3 gene defects in a wider range of overgrowth disorders.

The Wnt gene family has a role in development as well as tumourigenesis. One mouse member, Wnt7a, is vital for limb development in vivo and also possesses transforming ability in vitro. This study reports the isolation of a full length of human homologue of mouse Wnt7a gene by library screening. Yeast artificial chromosome-fluorescence in situ hybridisation (YAC-FISH) mapped the WNT7A gene to chromosome 3p25. Human WNT7A had an ORF encoding a deduced protein of 349 aa that exhibited 97% and 92% identity to mouse Wnt7a at the aa and nucleic acid levels, respectively. It possessed the 22 conserved cysteine residues and 3 more at the amino terminus, and a putative poly A tail. This is the fifth human WNT gene in which a complete cDNA sequence had been determined.

It is becoming increasingly clear that repetitive DNA is of biological significance as well as experimental importance. Here we review the information available about one type of repetitive DNA, the trinucleotide repeat (CAC)n, and briefly compare it with other trinucleotide repeats. Although much work has been done in analysing DNA fingerprinting patterns produced using the synthetic oligonucleotide (CAC)5 as a probe, there is relatively little information about individual (CAC)n-containing sequences and their abundance, organisation and distribution in mammalian DNA. From the data that is available, it is clear that there are at least two areas that should repay further study: (1) the organisation and generation of long sequences that contain (CAC)n motifs as part of a larger repeating unit (minisatellites) and (2) the distribution of small (CAC)n sequences (microsatellites), in particular their relationship to genes.

Three microsatellites have been identified in cosmids from the human X chromosome. The cosmids have been assigned locus numbers DXS554, DXS559, and DXS566 and have been localized to Xq12-q13 (DXS554 and DXS559) and Xq13 (DXS566). In addition, they have been genetically mapped in relation to the androgen receptor (AR), phosphoglycerate kinase 1, pseudogene 1 (PGK1P1), and phosphoglycerate kinase (PGK1) loci in the proximal long arm. Genetically, the localization of microsatellites at DXS554 and DXS566 is indistinguishable from PGK1, whereas that at DXS559 maps between AR and PGK1, close to PGK1P1. DXS566 is identical to the independently identified DXS441 marker. These markers should be useful for physical and genetic mapping in this region. 10 refs., 1 fig., 1 tab.

Four families, each with two individuals affectecd by Rett Syndrome (RS), were analysed using restriction fragment lenght polymorphisms and microsatellite markers from the X chromosome. In two of the families, X-linked dominant inheritance of the RS defect from a germinally mosaic mother could be assumed. Therefore, maternal X chromosome markers showing discordant inheritance were used to exclude regions of the X chromosome as locations of the RS gene. Much of the short arm could be excluded, including regions containing three candidate genes, OTC, synapsin 1 and synaptophysin. Although most of the long arm was inherited in common it was possible to exclude a centromeric region. Inheritance of X chromosome markers is also presented for two families with affected aunt-niece pairs, one of which has not been previously studied at the DNA level.

Aarskog syndrome has been mapped to Xq13 on the basis of a patient carrying an Xq13:8p21.2 translocation. We have identified a new microsatellite marker in a clone mapping to this region (HX60;DXS566). Using primers flanking this microsatellite along with primers detecting a microsatellite at PGK1P1 and DXS255, and DXS72, we have performed a multipoint analysis in a large kindred with Aarskog syndrome. Our results suggest that the Aarskog locus lies proximal to Xq13. This is supported by the recent redefining of the breakpoint of the original translocation as between DXS14 (Xp11.21-p11.1) and DXS146 (Xp11.23-p11.22).

There has recently been substantial progress in categorizing the vast range of human retinal degeneration phenotypes. A molecular approach has assigned chromosomal locations for approximately a dozen such diseases and has identified four of the genes involved.

A highly informative microsatellite marker, DXS426, which maps proximal to DXS7 in the interval Xp11.4-Xp11.23, has been used to refine further the localisation of the gene for Norrie disease (NDP). The results from a multiply informative crossover localize the NDP gene proximal to DXS7. In conjunction with information from 2 NDP patients who have a deletion for DXS7 but not for DSX426, our data indicate that the NDP gene lies between DXS7 and DXS426 on proximal Xp.

A CA repeat has been found on the human X chromosome within 16 kb of the gene encoding properdin P factor (PFC) and has been shown to be a highly informative marker. Two more polymorphic CA repeats were found in a cosmid containing DXS228. The CA repeats, and other markers from proximal Xp, were mapped genetically in CEPH families and the likely order of markers was established as Xpter-(DXS7, MAO-A, DXS228)-(PFC, DXS426)-(TIMP, OATL1)-DXS255-Xcen. This places PFC in the region Xp11.3–Xp11.23, thus refining previous in situ hybridization data. Two yeast artificial chromosomes (YACs) (440 and 390 kb) contain both PFC and DXS426, and one of them (440 kb) also contains TIMP. This confirms the genetic order TIMP-(PFC, DXS426). PFC and TIMP are located on the same 100-kb View the MathML source fragment of the 440-kb YAC. Given the genetic orientation of TIMP and (PFC, DXS426), this YAC can now serve as a starting point for directional walking toward disease genes located in Xp11.3–Xp11.2 such as retinitis pigmentosa (RP2) and Wiskott-Aldrich syndrome.

Summary Methylation of CCGG sites was examined in four regions of the X chromosome with four X-chromosome clones, three obtained by cloning random segments and one encoding a structural gene. In DNA from human peripheral blood cells unmethylated sites correlating with the inactive X chromosome were detected in the vicinity of two of the random clones and also in the vicinity of a cloned sequence of the X-linked phosphoglycerate kinase gene (PGK). The third random clone covered a region whose methylation pattern was unchanged between the active and inactive X chromosomes. Differential methylation at the sites detected appears to have no functional role in the maintenance of the inactive X chromosome since both active and inactive X chromosomes were found to be undermethylated in DNA from human lymphoblastoid cells.

We have determined the sequence of an 812-bp BamHI-EcoRI restriction fragment containing the 5' region of the human gene for PGK (3-phosphoglycerate kinase or ATP:3-phospho- d -glycerate 1-phosphotransferase; EC 2.7.2.3). The fragment contains 450 bp 5' to three start points for transcription (located by primer extension and S1 nuclease mapping), a leader sequence 85–94 bp long, the first exon of the gene (65 bp), and part of the first intron. The promoter region is extremely G + C-rich, lacks a TATA box, and has an 8-bp direct repeat. A comparison of the promoter region for PGK with other promoters on the X-chromosome reveals homology with the promoter for HPRT, but not with the operator for factor IX.

We report here the genetic cause of the X-linked syndrome of psychosis, pyramidal signs, and macro-orchidism (PPM-X) in a three-generation family manifesting the disorder as a mutation in the methyl-CpG binding–protein 2 (MECP2) gene in Xq28. The A140V mutation was found in all affected males and all carrier females in the family. To date, descriptions have been published of two patients with independent familial mental retardation (MR) and two patients with sporadic MR who harbor this specific mutation in the MECP2 gene. This strongly suggests that A140V is a hot spot of mutation resulting in moderate to severe MR in males. A simple and reliable PCR approach has been developed for detection of the hot spot A140V mutation to prescreen any other unexplained cases of MR before further extensive mutation analyses.

Only a small proportion of the vertebrate genome codes for proteins. It would therefore be useful if genes, and in particular the sites at which transcription begins, could be identified in libraries of cloned DNA. Since many known vertebrate genes have distinctive sequences (HTF-islands) surrounding their transcription start sites1, we wished to be able to select these sequences easily and to find out how diagnostic they are for genes. HTF-islands contain a high density of non-methylated CpG (ref. 2) and can be detected in chromosomal DNA as clustered sites for certain rare-cutting (C–G) restriction enzymes3. Identification of islands in chromosomal DNA is aided by methylation which blocks C–G enzyme sites in non-island DNA. This advantage is lost in cloned DNA, where CpG methylation is absent. We have calculated, however, that even in cloned DNA most sites for certain C–G enzymes should occur in HTF-islands. We tested this prediction using the enzyme SacII and found that four out of four sites in separate cosmids from the human X chromosome were located in HTF-islands. Hybridization to Northern blots provided preliminary evidence that three of the islands were associated with genes.

Methylation has been shown to be correlated with several fundamental cellular processes, including changes in gene expression, alterations in chromatin structure and inactivation of the mammalian X chromosome. It is possible, therefore, that the methylation status of a particular sequence may reflect involvement in a number of processes. Given this potentially confused situation, it is clear that many studies would be facilitated if unmethylated or minimally methylated DNA from a mammalian source were available. A major use of such DNA would be in the construction of long-range physical maps. In many cases, long-range physical maps are a prerequisite for the eventual isolation of disease genes that have been localised to a particular chromosomal region by other means (e.g. genetic linkage studies). Many of the enzymes used in such long-range mapping experiments are methylation sensitive, which makes it difficult to determine how many sites for a particular enzyme are present in any DNA sequence. Here we report the finding of a minimally methylated DNA in the human lymphoblastoid cell line, Til 1. The methylation level of Til 1 DNA was analysed in several ways and compared with that in human lymphocytes, placental tissue and other lymphoblastoid cell lines. The results showed clear and reproducible differences in methylation among the cell types, both at a global level and in the vicinities of specific DNA sequences. Lymphocyte DNA had the highest level of methylation while placental DNA and cell line DNA had lower but similar levels. Til 1 had abnormally low levels of 5-methylcytosine when measured directly, and no detectable methylation at any of the restriction sites examined.