Sharmila Banerjee-Basu

MLH3: a DNA mismatch repair gene associated with mammalian microsatellite instability.

DNA mismatch repair is important because of its role in maintaining genomic integrity and its association with hereditary non-polyposis colon cancer (HNPCC). To identify new human mismatch repair proteins, we probed nuclear extracts with the conserved carboxy-terminal MLH1 interaction domain. Here we describe the cloning and complete genomic sequence of MLH3, which encodes a new DNA mismatch repair protein that interacts with MLH1. MLH3 is more similar to mismatch repair proteins from yeast, plants, worms and bacteria than to any known mammalian protein, suggesting that its conserved sequence may confer unique functions in mice and humans. Cells in culture stably expressing a dominant-negative MLH3 protein exhibit microsatellite instability. Mlh3 is highly expressed in gastrointestinal epithelium and physically maps to the mouse complex trait locus colon cancer susceptibility I (Ccs1). Although we were unable to identify a mutation in the protein-coding region of Mlh3 in the susceptible mouse strain, colon tumours from congenic Ccs1 mice exhibit microsatellite instability. Functional redundancy among Mlh3, Pms1 and Pms2 may explain why neither Pms1 nor Pms2 mutant mice develop colon cancer, and why PMS1 and PMS2 mutations are only rarely found in HNPCC families.

AutDB: a gene reference resource for autism research

Recent advances in studies of Autism Spectrum Disorders (ASD) has uncovered many new candidate genes and continues to do so at an accelerated pace. To address the genetic complexity of ASD, we have developed AutDB (http://www.mindspec.org/autdb.html), a publicly available web-portal for on-going collection, manual annotation and visualization of genes linked to the disorder. We present a disease-driven database model in AutDB where all genes connected to ASD are collected and classified according to their genetic variation: candidates identified from genetic association studies, rare single gene mutations and genes linked to syndromic autism. Gene entries are richly annotated for their relevance to autism, along with an in-depth view of their molecular functions. The content of AutDB originates entirely from the published scientific literature and is organized to optimize its use by the research community. The main focus of this resource is to provide an up-to-date, annotated list of ASD candidate genes in the form of reference dataset for interrogating molecular mechanisms underlying the disorder. Our model for consolidated knowledge representation in genetically complex disorders could be replicated to study other such disorders.

Mutation of a gene encoding a putative chaperonin causes McKusick-Kaufman syndrome.

McKusick-Kaufman syndrome (MKKS, MIM 236700) is a human developmental anomaly syndrome comprising hydrometrocolpos (HMC), postaxial polydactyly (PAP) and congenital heart disease (CHD). MKKS has been mapped in the Old Order Amish population to 20p12, between D20S162 and D20S894 (ref. 3). Here we describe the identification of a gene mutated in MKKS. We analysed the approximately 450-kb candidate region by sample sequencing, which revealed the presence of several known genes and EST clusters. We evaluated candidate transcripts by northern-blot analysis of adult and fetal tissues. We selected one transcript with widespread expression, MKKS, for analysis in a patient from the Amish pedigree and a sporadic, non-Amish case. The Old Order Amish patient was found to be homozygous for an allele that had two missense substitutions and the non-Amish patient was a compound heterozygote for a frameshift mutation predicting premature protein truncation and a distinct missense mutation. The MKKS predicted protein shows amino acid similarity to the chaperonin family of proteins, suggesting a role for protein processing in limb, cardiac and reproductive system development. We believe that this is the first description of a human disorder caused by mutations affecting a putative chaperonin molecule.

Chicken homeobox gene prox 1 related to Drosophila prospero is expressed in the developing lens and retina

Prox 1 is the vertebrate homolog of Drosophila prospero, a gene known to be expressed in the lens‐secreting cone cells of fly ommatidia. Chicken Prox 1 cDNAs were isolated from 14 day embryonic chicken lenses, and a complete open reading frame encoding an 83 kDa protein was elucidated. The homeodomains of chicken and mouse Prox 1 are identical at the amino acid level and are 65–67% similar to the homeodomains of Drosophila and C. elegans prospero. The homology between these proteins extends beyond the homeodomain. There is 56% identity between chicken Prox 1 and Drosophila prospero in the C‐terminal region downstream of the homeodomain, whereas there is little similarity upstream of the homeodomain. Prox 1 is expressed most actively in the developing lens and midgut and at lower levels in the developing brain, heart, muscle, and retina. cDNA sequencing has established that there are alternatively spliced forms of the single Prox 1 gene, which probably account for the two abundant RNAs of about 2 and 8 kb and two less abundant RNAs close to 3.5 kb in length in the lens. In the lens fibers, only the shortest mRNA was present, whereas, in the epithelial cells, both short and long mRNAs were detected. By using in situ hybridization, expression of the Prox 1 gene was first detected at stage 14 in the early lens placode and slightly preceded the expression of δ1‐crystallin, the first crystallin gene expressed in the developing chicken lens. At later stages of development, Prox 1 mRNA was observed throughout the lens, but it appeared more abundant around the bow region of the equator than in the anterior epithelium or the fibers. In the retina, expression of the Prox 1 gene was detected mainly in the inner nuclear layer during later stages of histogenesis. The conserved pattern of Prox 1/prospero gene expression in vertebrates and Drosophila suggests that Prox 1, like Pax‐6, may be essential for eye development in different systematic groups.

Analyses of the effects that disease-causing missense mutations have on the structure and function of the winged-helix protein FOXC1.

Five missense mutations of the winged-helix FOXC1 transcription factor, found in patients with Axenfeld-Rieger (AR) malformations, were investigated for their effects on FOXC1 structure and function. Molecular modeling of the FOXC1 forkhead domain predicted that the missense mutations did not alter FOXC1 structure. Biochemical analyses indicated that, whereas all mutant proteins correctly localize to the cell nucleus, the I87M mutation reduced FOXC1-protein levels. DNA-binding experiments revealed that, although the S82T and S131L mutations decreased DNA binding, the F112S and I126M mutations did not. However, the F112S and I126M mutations decrease the transactivation ability of FOXC1. All the FOXC1 mutations had the net effect of reducing FOXC1 transactivation ability. These results indicate that the FOXC1 forkhead domain contains separable DNA-binding and transactivation functions. In addition, these findings demonstrate that reduced stability, DNA binding, or transactivation, all causing a decrease in the ability of FOXC1 to transactivate genes, can underlie AR malformations.

Loss-of-Function Mutations in Growth Differentiation Factor-1 (GDF1) Are Associated with Congenital Heart Defects in Humans

Congenital heart defects (CHDs) are among the most common birth defects in humans (incidence 8-10 per 1,000 live births). Although their etiology is often poorly understood, most are considered to arise from multifactorial influences, including environmental and genetic components, as well as from less common syndromic forms. We hypothesized that disturbances in left-right patterning could contribute to the pathogenesis of selected cardiac defects by interfering with the extrinsic cues leading to the proper looping and vessel remodeling of the normally asymmetrically developed heart and vessels. Here, we show that heterozygous loss-of-function mutations in the human GDF1 gene contribute to cardiac defects ranging from tetralogy of Fallot to transposition of the great arteries and that decreased TGF- beta signaling provides a framework for understanding their pathogenesis. These findings implicate perturbations of the TGF- beta signaling pathway in the causation of a major subclass of human CHDs.

The zebrafish gene claudinj is essential for normal ear function and important for the formation of the otoliths

We have identified a mutation in the zebrafish gene claudinj generated by retroviral integration. Mutant embryos display otoliths severely reduced in size, no response to tapping stimulus, and an inability to balance properly suggesting vestibular and hearing dysfunction. Antisense in situ hybridization to the cldnj gene showed expression first in the otic placode and later asymmetric expression in the otic vesicle. Morpholino inhibition of claudinj expression showed similar defects in otolith formation. Phylogenetic analysis of claudin sequences from multiple species demonstrates that claudinj was part of a gene expansion that began in the common ancestor of fish and humans, but additional fish specific gene duplications must have also occurred.

The Homeodomain Resource: 2003 update

The Homeodomain Resource is a searchable, curated collection of information for the homeodomain protein family. The resource is organized in a compact form and provides user-friendly interfaces for both querying the component databases and assembling customized datasets. The current release (version 5.0, October 2002) contains 1056 full-length homeodomain-containing sequences, 37 experimentally-derived structures, 81 homeodomain interactions, 84 homeodomain DNA-binding sites and 114 homeodomain proteins implicated in human genetic disorders. A new feature of this new release is the inclusion of experimentally-derived protein-protein interaction data for homeodomain family members. All entries are cross-linked for easy retrieval of the original records from source databases. The Homeodomain Resource is freely available through the World Wide Web at http://research.nhgri.nih.gov/homeodomain/.

The Homeodomain Resource: sequences, structures, DNA binding sites and genomic information

The Homeodomain Resource is an annotated collection of non-redundant protein sequences, three-dimensional structures and genomic information for the homeodomain protein family. Release 3.0 contains 795 full-length homeodomain-containing sequences, 32 experimentally-derived structures and 143 homeo-box loci implicated in human genetic disorders. Entries are fully hyperlinked to facilitate easy retrieval of the original records from source databases. A simple search engine with a graphical user interface is provided to query the component databases and assemble customized data sets. A new feature for this release is the addition of DNA recognition sites for all human homeodomain proteins described in the literature. The Homeodomain Resource is freely available through the World Wide Web at http://genome.nhgri.nih.gov/homeodomain.

The Homeodomain Resource: sequences, structures and genomic information

The Homeodomain Resource is a comprehensive collection of sequence, structure and genomic information on the homeodomain protein family. Available through the Resource are both full-length and domain-only sequence data, as well as X-ray and NMR structural data for proteins and protein-DNA complexes. Also available is information on human genetic diseases and disorders in which proteins from the homeodomain family play an important role; genomic information includes relevant gene symbols, cytogenetic map locations, and specific mutation data. Search engines are provided to allow users to easily query the component databases and assemble specialized data sets. The Homeodomain Resource is available through the World Wide Web at http://genome.nhgri.nih.gov/homeodomain