Thearith Koeuth

Homologous recombination of a flanking repeat gene cluster is a mechanism for a common contiguous gene deletion syndrome

Smith–Magenis syndrome (SMS), caused by del(17)p11.2, represents one of the most frequently observed human microdeletion syndromes. We have identified three copies of a low–copy–number repeat (SMS–REPs) located within and flanking the SMS common deletion region and show that SMS–REP represents a repeated gene cluster. We have isolated a corresponding cDNA clone that identifies a novel junction fragment from 29 unrelated SMS patients and a different–sized junction fragment from a patient with dup(17)p11.2. Our results suggest that homologous recombination of a flanking repeat gene cluster is a mechanism for this common microdeletion syndrome.

A recombination hotspot responsible for two inherited peripheral neuropathies is located near a mariner transposon-like element

The Charcot-Marie Tooth disease type 1A (CMT1 A) duplication and hereditary neuropathy with liability to pressure palsies (HNPP) deletion are reciprocal products of an unequal crossing-over event between misaligned flanking CMT1A-REP repeats. The molecular aetiology of this apparently homologous recombination event was examined by sequencing the crossover region. Through the detection of novel junction fragments from the recombinant CMT1 A-REPs in both CMT1A and HNPP patients, a 1.7-kb recombination hotspot within the ∼30-kb CMT1 A-REPs was identified. This hotspot is 98% identical between CMT1 A-REPs indicating that sequence identity is not likely the sole factor involved in promoting crossover events. Sequence analysis revealed a mariner transposon-like element (MITE) near the hotspot which we hypothesize could mediate strand exchange events via cleavage by a transposase at or near the 3′ end of the element.

DNA Rearrangements on Both Homologues of Chromosome 17 in a Mildly Delayed Individual with a Family History of Autosomal Dominant Carpal Tunnel Syndrome

Disorders known to be caused by molecular and cytogenetic abnormalities of the proximal short arm of chromosome 17 include Charcot-Marie-Tooth disease type 1A (CMT1A), hereditary neuropathy with liability to pressure palsies (HNPP), Smith-Magenis syndrome (SMS), and mental retardation and congenital anomalies associated with partial duplication of 17p. We identified a patient with multifocal mononeuropathies and mild distal neuropathy, growth hormone deficiency, and mild mental retardation who was found to have a duplication of the SMS region of 17p11.2 and a deletion of the peripheral myelin protein 22 (PMP22) gene within 17p12 on the homologous chromosome. Further molecular analyses reveal that the dup(17)(p11.2p11.2) is a de novo event but that the PMP22 deletion is familial. The family members with deletions of PMP22 have abnormalities indicative of carpal tunnel syndrome, documented by electrophysiological studies prior to molecular analysis. The chromosomal duplication was shown by interphase FISH analysis to be a tandem duplication. These data indicate that familial entrapment neuropathies, such as carpal tunnel syndrome and focal ulnar neuropathy syndrome, can occur because of deletions of the PMP22 gene. The co-occurrence of the 17p11.2 duplication and the PMP22 deletion in this patient likely reflects the relatively high frequency at which these abnormalities arise and the underlying molecular characteristics of the genome in this region.

Detection of the CMT1A/HNPP recombination hotspot in unrelated patients of European descent.

Charcot-Marie-Tooth type 1 disease (CMT1) and hereditary neuropathy with liability to pressure palsies (HNPP) are common inherited disorders of the peripheral nervous system. The majority of CMT1 patients have a 1.5Mb tandem duplication (CMT1A) in chromosome 17p11.2 while most HNPP patients have a deletion of the same 1.5 Mb region. The CMT1A duplication and HNPP deletion are the reciprocal products of an unequal crossing over event between misaligned flanking CMT1A-REP elements. We analysed 162 unrelated CMT1A duplication patients and HNPP deletion patients from 11 different countries for the presence of a recombination hotspot in the CMT1A-REP sequences. A hotspot for unequal crossing over between the misaligned flanking CMT1A-REP elements was observed through the detection of novel junction fragments in 76.9% of 130 unrelated CMT1A patients and in 71.9% of 32 unrelated HNPP patients. This recombination hotspot was also detected in eight out of 10 de novo CMT1A duplication and in two de novo HNPP deletion patients. These data indicate that the hotspot of unequal crossing over occurs in several populations independently of ethnic background and is directly involved in the pathogenesis of CMT1A and HNPP. We conclude that the detection of junction fragments from the CMT1A-REP element on Southern blot analysis is a simple and reliable DNA diagnostic tool for the identification of the CMT1A duplication and HNPP deletion in most patients.

Conservation and evolution of the rpsU‐dnaG‐rpoD macromolecular synthesis operon in bacteria

The macromolecular synthesis (MMS) operon contains three essential genes (rpsU, dnaG, rpoD) whose products (S21, primase, sigma‐70) are necessary for the initiation of protein, DNA, and RNA synthesis respectively. PCR amplifications with primers complementary to conserved regions within these three genes, and subsequent DNA sequencing of rpsU—dnaG PCR products, demonstrate that the three genes appear to be contiguous in 11 different Gram‐negative species. Within the Gram‐negative enteric bacterial lineage, the S21 amino acid sequence is absolutely conserved in 10 species examined. The putative nuteq antiterminator sequence in rpsU consists of two motifs, boxA and boxB, conserved in primary sequence and secondary structure. The terminator sequence, Ti, located between rpsU and dnaG is conserved at 31 positions in nine enterobacterial species, suggesting the importance of primary sequence in addition to secondary structure for transcription termination. The intergenic region between rpsU and dnaG varies in size owing to the presence or absence of the Enterobacterial Repetitive Intergenic Consensus (ERIC) DNA element. The rpoD gene contains rearrangements involving a divergent sequence, although two carboxy‐terminal regions which encode functional domains are conserved in primary sequence and spacing. Our data suggest that primary sequence divergence and DNA rearrangements in both coding and non‐coding sequences account for the interspecies variation in operon structure. However, MMS operon gene organization and cis‐acting regulatory sequences appear to be conserved in diverse bacteria.

Quality control for bacterial inhibition assays: DNA fingerprinting of microorganisms by rep-PCR

Abstract The bacterial inhibition assay (BIA) has been useful in a variety of newborn screening tests. BIAs rely on specific bacterial indicator strains as standardized components in different assays. Recently, DNA fingerprinting methods have been developed for eubacteria which utilize conserved repetitive DNA sequences. The repetitive DNA sequences, REP (Repetitive Extragenic Palindromic) and ERIC (Enterobacterial Repetitive Intergenic Consensus), can be used as oligonucleotide primer binding sites for the polymerase chain reaction (PCR) amplification of unique DNA sequences located between the repeats. Repetitive sequence based PCR, generally known as rep-PCR, allows the production of species- and strain-specific genomic fingerprint patterns following size-fractionation of the amplified DNA fragments by agarose gel electrophoresis. Two Bacillus subtilis strains, ATCC 6051 and 6633, currently used in newborn screening BlAs were distinguished by rep-PCR. Using rep-PCR to fingerprint bacterial strains collected from different newborn screening laboratories, an anomalous sample was revealed which yielded rep-PCR fingerprint patterns distinct from other laboratory samples of the same strain. Genomic fingerprinting by rep-PCR may be useful for quality control of specific bacterial strains used in BIAs.

Vertical transmission of Citrobacter diversus documented by DNA fingerprinting.

OBJECTIVE To confirm the vertical transmission of Citrobacter diversus from a mother to her infant and to evaluate the epidemiologic usefulness of a new automated procedure for analysis of polymerase chain reaction (PCR)-generated DNA fingerprints. DESIGN Repetitive element-based PCR (rep-PCR) analysis of C diversus isolates from the blood and amniotic fluid of a mother and the blood of her infant was performed. Unrelated C diversus isolates also were characterized and compared with the isolates from mother and infant. DNA fingerprints were generated by gel electrophoresis of PCR products derived from either unlabeled standard repetitive sequence-based oligonucleotide primers or fluorescent primers. The standard rep-PCR fingerprints were analyzed by visual inspection. The fluorescent primers were used in fluorophore-enhanced rep-PCR (FERP), and the FERP DNA fingerprints were analyzed by an Applied BioSystems (ABI) Model 373A laser scanning unit equipped with Genescan 672 software (Applied Biosystems, Inc, Foster City, CA). SETTING AND PATIENTS A mother and her newborn infant, both with invasive disease due to C diversus, in an urban tertiary-care hospital. RESULTS The DNA fingerprints of the maternal blood, amniotic fluid, and infant blood isolates of C diversus were identical by both visual inspection of ethidium bromide-stained agarose gels and computer-aided analysis of FERP patterns. These strains appeared to differ from all but one control isolate, which had been collected 7 years earlier in the same city in which the infant was born. CONCLUSIONS Vertical transmission of C diversus from mother to infant can occur in utero. Automated analysis of rep-PCR-generated DNA fingerprints derived using fluorescent primers is an objective means for comparing isolates of C diversus and in all likelihood would be useful for other species of bacteria that possess repetitive elements.

SERE, a widely dispersed bacterial repetitive DNA element

The presence of a Salmonella serotype Enteritidis repeat element (SERE) located within the upstream regulatory region of the sefABCD operon encoding fimbrial proteins is reported. DNA dot-blot hybridisation analyses and computerised searches of genetic databases indicate that SERE is well conserved and widely distributed throughout the bacterial and archaeal kingdoms. A SERE-based polymerase chain reaction (SERE-PCR) assay was developed to fingerprint 54 isolates of Enteritidis representing nine distinct phage types and 54 isolates of other Salmonella serotypes. SERE-PCR identified five distinct fingerprint profiles among the 54 Enteritidis isolates; no correlation between phage types and SERE-PCR fingerprint patterns was noticed. SERE-PCR was reproducible, rapid and easy to perform. The results of this investigation suggest that the limited heterogeneity of SERE-PCR fingerprint patterns can be utilised to develop serotype- and serogroup-specific fingerprint patterns for isolates of Enteritidis.

RAPID TYPING OF NEISSERIA MENINGITIDIS ISOLATES BY REPETITIVE ELEMENT-BASED PCR (rep-PCR) DNA FINGERPRINTING. † 1115

RAPID TYPING OF NEISSERIA MENINGITIDIS ISOLATES BY REPETITIVE ELEMENT-BASED PCR (rep-PCR) DNA FINGERPRINTING. † 1115