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PubMed2005

Microbial enzymes mined from the Urania deep-sea hypersaline anoxic basin.

We created a metagenome expression library from the brine:seawater interface of the Urania hypersaline basin, screened it for esterases, and characterized five of these. Two had no significant sequence homology to known esterases, hydrolyzed both carboxylesters and thioesters, and exhibited unusual, habitat-specific characteristics (preference for high hydrostatic pressure and salinity). One has an unusual structural signature incorporating three catalytic active centers mediating distinct hydrolytic activities and an adaptive tertiary-quaternary structure that alters between three molecular states, according to the prevailing physicochemical conditions. Some of the esterases have high activities, specificities, enantioselectivities, and exceptional stability in polar solvents, and they are therefore potentially useful for industrial biotransformations. One possesses the highest enantioselectivity toward an ester of the important chiral synthon solketal (E: 126[S]; 98%ee).

Bacteria, Anaerobic↗
PubMed1998

Lower-limb deep venous thrombosis: direct MR imaging of the thrombus.

PURPOSE: To determine the feasibility of diagnosing deep venous thrombosis (DVT) with direct three-dimensional magnetic resonance (MR) imaging of the thrombus. MATERIALS AND METHODS: Eighteen patients with proved DVT at conventional venography were evaluated with a magnetization-prepared three-dimensional blood- and fat-suppressed MR imaging sequence. The presence and extent of thrombosis on the MR images were compared with the findings at conventional venography. RESULTS: At MR imaging, DVT was visualized in 17 of the 18 patients. MR imaging demonstrated greater proximal extent of the thrombosis in four patients, asymptomatic contralateral thrombosis in one, involvement of the deep femoral vein in five, and involvement of the superficial venous system in three. CONCLUSION: Direct MR imaging of clots appears capable of demonstrating venographically diagnosed DVT. This MR imaging technique is noninvasive, quick, and repeatable and allows a survey of the whole lower-limb venous system. Detection of thrombosis relies on the formation of methemoglobin, which appears to be sufficiently rapid to allow use of this technique in the clinical setting. Changes in the signal from clot over time may allow estimation of the age of the thrombus.

Feasibility Studies↗
PubMed1997

[Determination of cartilage thickness in the ankle joint. an MRT (1.5)-anatomical comparative study].

PURPOSE: The aim of this study was to evaluate the accuracy of MRI in the measurement of cartilage thickness of the ankle joint in comparison to pathologic and histologic specimens. PATIENTS AND METHODS: The ankle joints of four fresh cadaver feet were imaged on a 1.5T MR-unit in the coronal plane. Standard T1-weighted spin echo (SE) and a T1-weighted 3D-GE (FLASH-3D) sequence with fat saturation were applied. Following MR imaging, the talus was explanted and cut parallel to the MR images for macroscopic evaluation and histologic specimens were prepared. MRI measurements of the cartilage thickness of the talus were carried out in two ways: without and with consideration of a transition zone of intermediate signal intensity between hyperintense cartilage and hypointense cortical bone. The data were compared with the anatomic specimens as the gold standard expressing the difference as a percentage value. On histologic specimens thickness of deep calcified cartilage layer was measured. RESULTS: Measurements without the transition zone showed a mean underestimation of talus cartilage thickness of 46.8% (41.3-52.1) for T1-SE and 47.5% (43.1-52.1) for fat saturated FLASH-3D images. Considering the transition zone the mean values were 25.0% (23.1-26.2) and 14.1% (6.7-21.5). The histologic specimens showed a three-fold increase of thickness of deep calcified cartilage layer. CONCLUSIONS: Measurements of the cartilage layer of the ankle joint on MR images are only accurate if the transition zone (calcified cartilage layer) is considered and the optimal pulse sequence (FLASH-3D-fat-sat) is applied.

Aged↗
PubMed2000

Diversity and community structure within anoxic sediment from marine salinity meromictic lakes and a coastal meromictic marine basin, Vestfold Hilds, Eastern Antarctica.

16S rDNA clone library analysis was used to examine the biodiversity and community structure within anoxic sediments of several marine-type salinity meromictic lakes and a coastal marine basin located in the Vestfolds Hills area of Eastern Antarctica. From 69 to 130 (555 total) 16S rDNA clones were analysed from each sediment sample, and restriction fragment length polymorphism (RFLP) and sequence analysis grouped the clones into 202 distinct phylotypes (a clone group with sequence similarity of >0.98). A number of phylotypes and phylotype groups predominated in all libraries, with a group of 10 phylotypes (31% of clones) forming a novel deep branch within the low G+C Gram-positive division. Other abundant phylotypes detected in several different clone libraries grouped with Prochlorococcus cyanobacteria, diatom chloroplasts, delta proteobacteria (Desulfosarcina group, Syntrophus and Geobacterl Pelobacter/Desulphuromonas group), order Chlamydiales (Parachlamydiaceae) and Spirochaetales (wall-less Antarctic spirochaetes). Most archaeal clones detected (3.1% of clones) belonged to a highly diverged group of Euryarchaeota clustering with clones previously detected in rice soil, aquifer sediments and hydrothermal vent material. Little similarity existed between the phylotypes detected in this study and other clone libraries based on marine sediment, suggesting that an enormous prokaryotic diversity occurs within marine and marine-derived sediments.

Antarctic Regions↗
PubMed2001

Higher-level phylogeny of new world vireos (aves: vireonidae) based on sequences of multiple mitochondrial DNA genes.

Interfamilial relationships of the New World songbird family Vireonidae are uncertain. Thus, we sequenced 3069 bp of four mitochondrial genes (cyt b, ND2, ND3, COI) from 19 taxa in five families and two outgroups, to examine higher-level alliances with proposed relatives. We also sequenced cyt b and ND2 from an additional five vireonids to examine intergeneric relationships within the Vireonidae and incorporated 14 sequences of cyt b from GenBank to test the effects of taxon sampling on gene tree resolution. Families appeared monophyletic in all analyses, and the affinity of vireonids to Old World corvoids was corroborated. However, relationships among the Vireonidae and other families were not resolved. Sequences of vireonids revealed high levels of divergence within and between genera, with either Cyclarhis or Vireolanius positioned basally, depending on the analysis. On the basis of mitochondrial DNA and biogeographic evidence, vireonids represent a deep lineage derived from an Old World ancestor that colonized the New World, most likely via Beringia, with subsequent radiation in the Middle American tropics. We hypothesize postcolonization dispersal of the ancestor into Middle America, followed by extinction of the ancestor in North America. This extinction event left the North Temperate Zone unoccupied by any vireonid until northward reinvasion by some species of Vireo. Although the closest living relative of vireonids remains unidentified, broad-scale sequencing of additional extant corvoids with multiple molecular markers should further elucidate Old World alliances.

Animals↗
PubMed2000

Phylogeny and biogeography of serolid isopods (Crustacea, Isopoda, Serolidae) and the use of ribosomal expansion segments in molecular systematics.

In this study, a molecular phylogenetic hypothesis for 16 species of serolid isopods (Crustacea, Isopoda, Serolidae) from Antarctic waters, the deep sea, South America, and Australia is presented. The genes used are a 500-bp fragment of the mitochondrial LSU rRNA gene and a 700-bp fragment located in the variable region V4 of the nuclear SSU rRNA gene. The species composition and monophyly of morphologically defined genera of which several members were available are confirmed by the molecular data (Ceratoserolis, Spinoserolis, and Cuspidoserolis). The molecular data also support the redefinition of Frontoserolis s.l. and Serolella and the erection of the new genera Septemserolis and Paraserolis, as proposed by W]agele. The relationship among several genera is resolved differently in the molecular hypothesis than in the two existing morphological hypotheses, however. The molecular phylogeny may have important consequences for understanding the biogeography of the Serolidae, indicating that all Antarctic species in this study form a monophyletic group which has probably derived from species with closest extant relatives in South America. All 3 species included in this study living today in deep waters (>2000 m) of the Southern Ocean are most closely related to species living on the Antarctic shelf, so that parallel colonization of the deep sea by way of polar submergence can be reconstructed. In this study, a V4 expansion segment is reported which exceeds the longest crustacean sequences known until now by more than 270 bp. Although the V4 expansion segment has proven useful for phylogenetic purposes in this study, there is circumstantial evidence that its mechanism of evolution may depend not only on inheritance of single-site substitutions, making its routine use in phylogenetic studies potentially dangerous.

Animals↗
PubMed2001

Mosses share mitochondrial group II introns with flowering plants, not with liverworts.

Extant bryophytes are regarded as the closest living relatives of the first land plants, but relationships among the bryophyte classes (mosses, liverworts and hornworts) and between them and other embryophytes have remained unclear. We have recently found that plant mitochondrial genes with positionally stable introns are well suited for addressing questions of plant phylogeny at a deep level. To explore further data sets we have chosen to investigate the mitochondrial genes nad4 and nad7, which are particularly rich in intron sequences. Surprisingly, we find that in these genes mosses share three group II introns with flowering plants, but none with the liverwort Marchantia polymorpha or other liverworts investigated here. In mitochondria of Marchantia, nad7 is a pseudogene containing stop codons, but nad7 appears as a functional mitochondrial gene in mosses, including the isolated genus Takakia. We observe the necessity for strikingly frequent C-to-U RNA editing to reconstitute conserved codons in Takakia when compared to other mosses. The findings underline the great evolutionary distances among the bryophytes as the presumptive oldest division of land plants. A scenario involving differential intron gains from fungal sources in what are perhaps the two earliest diverging land plant lineages, liverworts and other embryophytes, is discussed. With their positionally stable introns, nad4 and nad7 represent novel marker genes that may permit a detailed phylogenetic resolution of early clades of land plants.

Amino Acid Sequence↗
PubMed1984

Development of GABAergic neurons in rat visual cortex as identified by glutamate decarboxylase-like immunoreactivity.

The development of glutamate decarboxylase (GAD)-like immunoreactivity was studied in albino rats. GAD-positive structures appeared and accumulated in a characteristic temporo-spatial sequence: from embryonic day 18 onwards in lamina (L) I and subplate; after birth in L I-II, L IV-V and deep L VI including white matter; after two weeks in superficial L I, L II to L III-IV border, L V and L VI-white matter. GAD-positive axon varicosities formed perisomatic 'baskets' on L V pyramids on postnatal day 8 and a few days later on those L II-III. During the third week GAD-positive structures attained adult densities and established different laminar patterns in the primary and secondary visual areas.

Animals↗
PubMed2004

Structure and functions of the endothelial cell protein C receptor.

The endothelial cell protein C receptor (EPCR) plays a critical role in augmenting protein C activation by the thrombin-thrombomodulin complex and in modulating the functions of the protein C pathway to aid in preventing organ damage due to various challenges. EPCR exhibits a sequence and three-dimensional homology with the major histocompatibility class 1/CD1 family of proteins. This family of proteins is characterized by having a deep groove that is usually used in antigen presentation. In the case of CD1c and CD1d, this groove is filled with a lipid antigen, usually a glycolipid. Like the CD1 series, EPCR has a lipid in the corresponding groove. In this case, the lipid is usually phosphatidylcholine, but it may be phosphatidylethanolamine. The bound lipid contributes to protein C binding, but its structure suggests a role in maintaining EPCR structure rather than contributing directly to protein C binding. Potential roles for EPCR in hematopoiesis are suggested by the finding that EPCR is located on hematopoietic stem cells at reasonably high concentrations. The structure and the lipid antigen suggest that EPCR may be involved in preventing autoimmunity, which would be consistent with findings in CD1d knockout mice. Complete deletion of EPCR function results in embryonic death, at least in part due to placental thrombosis. In adult animals, the anticoagulant and anti-inflammatory responses to endotoxin increase with increasing EPCR expression. Some of the anti-inflammatory activity is likely to be due to EPCR's interactions with the integrin Mac-1 (CD11b/CD18) on leukocytes, an interaction that probably limits tight adhesion of leukocytes to activated endothelium. Thus, available data suggest a potential role of EPCR in hematopoiesis, autoimmunity, and the control of both the coagulation and inflammation responses to infection and trauma.

Animals↗
PubMed2005

African endemics span the tree of songbirds (Passeri): molecular systematics of several evolutionary 'enigmas'.

The deep divergence between the African endemic passerines Picathartidae (rockfowl Picathartes and rockjumpers Chaetops, four species) and the Passerida (ca. 3500 species) suggests an older history of oscines on the African continent than has previously been assumed. In order to determine whether any additional, unexpectedly deep lineages occur in African endemic songbirds, 29 species--including 10 enigmatic focal taxa endemic to southern Africa--were added to a large nuclear sequence dataset gathered from oscine songbirds (Passeri). Phylogenetic analyses of these data resolve many long-standing questions about the affinities of these birds, not all of which were predicted by traditional approaches. The application of a molecular clock indicates that most basal divergences in Passerida occurred in the middle to late Eocene, with divergences between African and Australasian core corvoids occurring somewhat later in the early Miocene. Consistent with inferences for mammals, divergences between Malagasy endemic passerines and their mainland relatives suggests an asynchronous colonization history. This emerging phylogenetic picture reveals that relationships within Old World families are highly informative regarding the early dispersal and radiation of songbirds out of Gondwana. Future analyses will depend on improving resolution of higher-level phylogenetic relationships among these groups, and increasing the density of taxon sampling within them.

Africa↗
PubMed2002

Direct quantitative differentiation between Prevotella intermedia and Prevotella nigrescens in clinical specimens.

This paper describes a quantitative fluorescent in situ hybridization (FISH) assay for the differential identification of Prevotella intermedia and Prevotella nigrescens in clinical samples, and compares its performance with less discriminatory culture and quantitative immunofluorescence (IF) assays. Fluorescence-labelled oligonucleotide probes directed to specific 16S rRNA sequences of P. intermedia, P. nigrescens, Prevotella pallens and Prevotella denticola were hybridized under stringent conditions with cultured reference strains or plaque samples from deep periodontal pockets. Probe specificity was defined with strains from multiple oral Prevotella species. The lower detection level of the assays was approximately 3x10(3) target cells per ml of plaque-sample suspension. P. intermedia, P. nigrescens, P. pallens and P. denticola were detected in plaques with prevalences of 69, 67, 0 and 28%, respectively. On average, 3.9 x 10(6) P. intermedia, 3.1 x 10(6) P. nigrescens and 5.6 x 10(5) P. denticola cells were counted per positive sample. All three species were found almost exclusively in dense mixed aggregates. Quantitative FISH data agreed satisfactorily with corresponding IF data (r=0.711). Both FISH and IF enumerations of the sum of P. intermedia and P. nigrescens markedly exceeded the c.f.u. counts of black-pigmented colonies in Porphyromonas gingivalis-free cultured subgingival plaques. The results demonstrate the validity of this new assay. Unlike established IF, culture, PCR or checkerboard DNA hybridization assays, this FISH assay differentiates quantitatively between P. intermedia and P. nigrescens, provides visual accuracy control, and offers insights into the spatial distribution of the target cells within a clinical sample.

Bacterial Infections↗
PubMed2006

Differentiation of sCJD and vCJD forms by automated analysis of basal ganglia intensity distribution in multisequence MRI of the brain--definition and evaluation of new MRI-based ratios.

We present a method for the analysis of basal ganglia (including the thalamus) for accurate detection of human spongiform encephalopathy in multisequence magnetic resonance imaging (MRI) of the brain. One common feature of most forms of prion protein diseases is the appearance of hyperintensities in the deep grey matter area of the brain in T2-weighted magnetic resonance (MR) images. We employ T1, T2, and Flair-T2 MR sequences for the detection of intensity deviations in the internal nuclei. First, the MR data are registered to a probabilistic atlas and normalized in intensity. Then smoothing is applied with edge enhancement. The segmentation of hyperintensities is performed using a model of the human visual system. For more accurate results, a priori anatomical data from a segmented atlas are employed to refine the registration and remove false positives. The results are robust over the patient data and in accordance with the clinical ground truth. Our method further allows the quantification of intensity distributions in basal ganglia. The caudate nuclei are highlighted as main areas of diagnosis of sporadic Creutzfeldt-Jakob Disease (sCJD), in agreement with the histological data. The algorithm permitted the classification of the intensities of abnormal signals in sCJD patient FLAIR images with a higher hypersignal in caudate nuclei (10/10) and putamen (6/10) than in thalami. Defining normalized MRI measures of the intensity relations between the internal grey nuclei of patients, we robustly differentiate sCJD and variant CJD (vCJD) patients, in an attempt to create an automatic classification tool of human spongiform encephalopathies.

Algorithms↗
PubMed1998

Phylogeny and identification in situ of Nevskia ramosa.

An enrichment of the neuston bacterium Nevskia ramosa was investigated by the cultivation-independent rRNA approach. N. ramosa was first described by Famintzin in 1892 as a rod-shaped, slightly bent bacterium forming typical flat rosettes on the surface of shallow freshwater habitats by unilateral slime formation. PCR in combination with cloning and sequencing was used for retrieving 21 partial and 5 nearly full-length 16S rRNA sequences forming three tight clusters. In situ hybridization with rRNA-targeted oligonucleotide probes allowed us to assign the three sequence clusters to three distinct bacterial populations abundant in the enrichment. The two probes that unambiguously identified the N. ramosa morphotype were derived from a 16S rRNA sequence that had similarities of 87.9 to 88.9% to the rRNA sequences of the most closely related group in the database, Xanthomonas sp. and relatives. N. ramosa currently is the only representative of an independent, deep branch of the gamma subclass of the class Proteobacteria. The two other populations abundant in the enrichment were affiliated with the alpha subclass of the class Proteobacteria. They were most closely related to Blastobacter sp. (97.2% similarity) and Mycoplana bullata (97.6% similarity) and might represent new species in the respective genera.

Bacteria↗
PubMed2006

Brain lesions on MRI in elderly patients with type 2 diabetes mellitus.

BACKGROUND AND PURPOSE: Diabetes mellitus (DM) type 2 has been associated with poor cognitive performance and dementia, particularly in elderly patients. The exact mechanisms underlying the cognitive dysfunction in DM remain unclear. Imaging studies of the brain could be helpful to give more insight into possible structural brain lesions underlying these cognitive dysfunctions. Therefore, we performed a study in independently living patients with DM type 2 in order to investigate the association between DM and brain imaging abnormalities. METHODS: The study population consisted of 45 patients with DM type 2 without hypertension (mean age 73.4 +/- 5.1 years, mean duration 16.5 +/- 11.5 years), 45 patients with DM type 2 and hypertension (mean age 73.5 +/- 6.1 years, mean duration 11.9 +/- 9.2 years) and 44 control subjects (mean age 73.1 +/- 5.4 years). All patients and control subjects underwent an MRI of the brain. White matter lesions (WML), cerebral atrophy and medial temporal lobe atrophy were rated by a standardized visual rating scale. Lacunar infarcts were defined as focal hypo-intensities on fluid-attenuated inversion recovery sequences with a hyperintense rim around it. RESULTS: WML occurred more frequently in diabetic patients with hypertension as well as without hypertension. Significantly more deep WML were found in DM patients with and without hypertension when compared to control subjects, whereas no difference was found in the occurrence of periventricular hyperintensities. In all 3 groups, lacunar infarcts occurred sporadically. A trend towards higher atrophy scores was seen in patients with DM compared to control subjects. CONCLUSIONS: The data of this cross-sectional study suggest that type 2 DM is an independent risk factor for deep WML in the independently living elderly patients.

Aged↗
PubMed2005

Severe Staphylococcal sepsis in adolescents in the era of community-acquired methicillin-resistant Staphylococcus aureus.

OBJECTIVE: More than 70% of the community-acquired (CA) staphylococcal infections treated at Texas Children's Hospital are caused by methicillin-resistant Staphylococcus aureus (MRSA). Since September 2002, an increase in the number of severely ill patients with S aureus infections has occurred. This study provides a clinical description of severely ill adolescent patients and an analysis of their isolates using molecular methods. METHODS: We identified adolescent patients meeting criteria for severe sepsis requiring admission to the PICU. Patient records were reviewed, and isolates were obtained for susceptibility testing and DNA extraction. Isolates were tested for the presence of virulence genes (cna, tst, lukS-PV, and lukF-PV) and enterotoxin genes (sea, seb, sec, sed, seh, and sej) by polymerase chain reaction. Genomic fingerprints were determined by repetitive-element polymorphism polymerase chain reaction and pulse-field gel electrophoresis. SCCmec cassette type was determined. RESULTS: Fourteen adolescents with severe CA S aureus infections were identified between August 2002 and January 2004. All were admitted to the PICU with sepsis and coagulopathy. Twelve patients had CA-MRSA infections; 2 had CA methicillin-susceptible Staphylococcus aureus (MSSA) infections. The mean age was 12.9 years (range: 10-15 years). Thirteen patients had pulmonary involvement and/or bone and joint infection; 10 patients had > or =2 bones or joints infected (range: 2-10); 4 patients developed vascular complications (deep venous thrombosis); and 3 patients died. All isolates were identical or closely related to the previously reported predominant clone in Houston, Texas (multilocus sequence type 8, USA300), and carried lukS-PV and lukF-PV genes as well as the SCCmec type IVa cassette (12 MRSA isolates) but did not contain cna or tst. Only 1 strain carried enterotoxin genes (sed and sej). CONCLUSIONS: Severe staphylococcal infections in previously healthy adolescents without predisposing risk factors have presented more frequently at Texas Children's Hospital since September 2002. CA MRSA and clonally related CA MSSA characterized as USA300 and sequence type 8 have been isolated from these patients.

Adolescent↗
PubMed2006

Structural basis for the inactivation of AdoMetDC K12R mutant.

S-adenosylmethionine decarboxylase (AdoMetDC) is a key enzyme in the biosynthesis of the polyamines spermidine and spermine. Polyamines are ubiquitous organic cations that are absolutely required for normal cell proliferation and differentiation. AdoMetDC catalyzes decarboxylation of S-adenosylmethionine (AdoMet) which provides aminopropyl groups for spermidine and spermine synthesis. Mammalian AdoMetDC is produced as a proenzyme (38 kDa) which is cleaved to form the alpha (30.7 kDa) and beta (7.7 kDa) subunits of the mature enzyme. It is here shown that the catalytic activity of the enzyme was completely eliminated when lysine 12 was mutated to an arginine residue in the small subunit; however, the proenzyme processing was not affected. On the other hand, mutations of other lysine residues (Lys45-->Arg and Lys56-->Arg) did not affect either the enzyme activity or the proenzyme processing. Structure analysis using Swiss Deep Viewer v3.7 has indicated that Arg in place of Lys12 may eliminate AdoMetDC activity by restricting the mobility of Thr85 through hydrogen bonding. Sequence alignment of various AdoMetDC sequences indicated that Thr85 is in a highly conserved region, suggesting that Thr85 is critical for the decarboxylation reaction.

Adenosylmethionine Decarboxylase↗
PubMedDate not supplied

[Acute myelopathy in a diver caused by decompression sickness. A case description and a survey of the literature].

INTRODUCTION: Decompression sickness (DS) is caused when bubbles of an inert gas usually nitrogen, since oxygen is metabolised in the tissues are released into the bloodstream and tissues during fast ascents once the atmospheric pressure is lowered near the surface. Neurological complications are its most serious form of expression and include vertigo, headache, stroke and acute myelopathy, among others. DS that affects the spinal cord is infrequent. CASE REPORT: A male, 42 years old, who presented progressive tetraparesis 15 minutes after returning to the surface following several immersions up to 40 metres deep in the same day. Neurological exploration revealed tetraparesis that was predominantly distal and in the lower limbs, a posterior cord syndrome, urinary incontinence and neurogenic pain. Total column magnetic resonance imaging showed areas of diffused hypersignal in the T2 sequence in the thoracic and cervical (C2 to C6) regions, predominating in the posterior cords. The echocardiogram, transcranial Doppler and spirometric studies ruled out an arterial gas embolism following pulmonary barotrauma. CONCLUSIONS: Spinal DS can give rise to a serious myelopathy, which affects the pyramidal pathway, posterior cords and sphincteral control, and which generally appears after sudden ascents from the deep dives.

Adult↗
PubMed2006

[A DNA duplication at chromosome 10q24.3 is associated with split-hand split-foot malformation in a Chinese family].

OBJECTIVE: To identify the disease-causing genetic alteration of split-hand/split-foot malformation (SHFM) in a Chinese family. METHODS: Three of the 5 affected individuals from a four-generation Chinese SHFM family were examined physically and radiologically. Peripheral blood samples were collected from Digital photographs of the malformed hands and feet were taken. Peripheral blood samples were collected from 2 affected individuals, and lymphocytes were isolated to undergo high resolution G-banding. Genomic DNA was extracted from the whole blood samples of 4 available family members, including the 3 affected individuals. All 16 exons and their flanking intronic sequences of the TP63 gene were amplified using polymerase chain reaction (PCR) and sequenced directly. Microsatellite markers from the five SHFM loci were analyzed in the available family members by PCR, polyacrylamide gel electrophoresis and silver staining. For semi-quantitative determination of the allele copy number, the polymorphic PCR-amplified fragments representing genetic markers from the SHFM3 locus at chromosome 10q24.3 were sequenced in the affected individuals using normal individuals with identical genotypes as controls. RESULTS: All 3 existing affected individuals showed absence of 3 radial fingers, 2 affected individuals had a deep central cleft and central ray deficiency in the feet, and 1 affected individual had a fibular monodactyli, all limb malformations being bilateral and consistent with the phenotype of typical SHFM. G-banding showed normal karyotypes in the 3 affected individuals and no visible cytogenetic abnormality was found. Moreover, no mutation was identified in the TP63 gene. While no haplotype sharing was observed in the markers from loci SHFM1, SHFM4 and SHFM5, potential haplotype sharing was detected in the markers from two loci, SHFM2 and SHFM3, indicating possible causative mutation at SHFM2 or SHFM3. Furthermore, obviously biased silver density toward the allele fragments shared by the 3 affected individuals was observed in the markers from the SHFM3 locus. Comparative sequencing showed roughly one-fold increase of fluorescent signal of the shared fragments in the affected individuals. These results suggested a large-scale DNA duplication within the SHFM3 locus. CONCLUSION: A large-scale DNA duplication within the SHFM3 locus at chromosome 10q24.3 has been identified as the pathogenic genetic change in Chinese patients with SHFM.

Base Sequence↗