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We expand the Kosaki overgrowth syndrome (KOGS) phenotype by over 70% to include 24 unreported KOGS symptoms, in a first male patient, the third overall associated with the PDGFRB c.1751C>G p.(Pro584Arg) mutation. Eighteen of these symptoms are unique to our patient, the remaining six are shared with other patients. Of the 24 unreported features overall, 6 show marked phenotype evolution and varying time of onset. The triangular face detected at 14 months and long palpebral fissures with lateral ectropion at 4 years are present in other members of the cohort. The remaining 4 are unique to Patient 5: pronounced macrocephaly from birth, increasingly triangular anterior skull from 14 months, camptodactyly, emerging at 4 years and worsening joint contractures from 6 years. Compilation of all new symptoms reported here with published clinical data further identifies at least 18 clinical parameters common to all cases to date, encompassing both known KOGS-associated PDGFRB mutations. We therefore propose a set of 18 core KOGS symptoms, with 16 present in early childhood. These results should also impact diagnostic/prognostic scope, intervention and outcome potential for KOGS patients, particularly for developmentally progressive conditions such as scoliosis and myofibroma.

Tooth agenesis is a common craniofacial abnormality in humans and represents failure to develop 1 or more permanent teeth. Tooth agenesis is complex, and variations in about a dozen genes have been reported as contributing to the etiology. Here, we combined whole-exome sequencing, array-based genotyping, and linkage analysis to identify putative pathogenic variants in candidate disease genes for tooth agenesis in 10 multiplex Turkish families. Novel homozygous and heterozygous variants in LRP6, DKK1, LAMA3, and COL17A1 genes, as well as known variants in WNT10A, were identified as likely pathogenic in isolated tooth agenesis. Novel variants in KREMEN1 were identified as likely pathogenic in 2 families with suspected syndromic tooth agenesis. Variants in more than 1 gene were identified segregating with tooth agenesis in 2 families, suggesting oligogenic inheritance. Structural modeling of missense variants suggests deleterious effects to the encoded proteins. Functional analysis of an indel variant (c.3607+3_6del) in LRP6 suggested that the predicted resulting mRNA is subject to nonsense-mediated decay. Our results support a major role for WNT pathways genes in the etiology of tooth agenesis while revealing new candidate genes. Moreover, oligogenic cosegregation was suggestive for complex inheritance and potentially complex gene product interactions during development, contributing to improved understanding of the genetic etiology of familial tooth agenesis.

Williams-Beuren syndrome (WBS) is a multisystemic genomic disorder typically caused by a recurrent ˜1.5-1.8 Mb deletion on 7q11.23. Atypical deletions can provide important insight into the genotype-phenotype correlations. Here, we report the phenotypic and molecular characterization of a girl with a de novo 81.8 kb deletion in the WBS critical region, which involves the ELN and LIMK1 genes only. The patient presented at 2 months of age with extensive vascular abnormalities, mild facial dysmorphism and delays in her fine motor skills. We discuss potential molecular mechanisms and the role of ELN and LIMK1 in the different phenotypic features. We compare the findings in our patient with previously reported overlapping deletions. The phenotypic variability among these patients suggests that other factors are important in the phenotype and possibly include: position effects related to copy number variation size, variations in the non-deleted alleles, genetic modifiers elsewhere in the genome, or reduced penetrance for specific phenotypes.

The advent of whole-exome next-generation sequencing (WES) has been pivotal for the molecular characterization of Mendelian disease; however, the clinical applicability of WES has remained relatively unexplored. We describe our exploration of WES as a diagnostic tool in a 3½-year old female patient with a 2-year history of episodic muscle weakness and paroxysmal dystonia who presented following a previous extensive but unrevealing diagnostic work-up. WES was performed on the proband and her two parents. Parental exome data was used to filter potential de novo genomic events in the proband and suspected variants were confirmed using di-deoxy sequencing. WES revealed a de novo non-synonymous mutation in exon 21 of the calcium channel gene CACNA1S that has been previously reported in a single patient as a rare cause of atypical hypokalemic periodic paralysis. This was unexpected, as the proband's original differential diagnosis had included hypokalemic periodic paralysis, but clinical and laboratory features were equivocal, and standard clinical molecular testing for hypokalemic periodic paralysis and related disorders was negative. This report highlights the potential diagnostic utility of WES in clinical practice, with implications for the approach to similar diagnostic dilemmas in the future.

Deletions in the 17p13.3 region are associated with abnormal neuronal migration. Point mutations or deletion copy number variants of the PAFAH1B1 gene in this genomic region cause lissencephaly, whereas extended deletions involving both PAFAH1B1 and YWHAE result in Miller-Dieker syndrome characterised by facial dysmorphisms and a more severe grade of lissencephaly. The phenotypic consequences of YWHAE deletion without deletion of PAFAH1B1 have not been studied systematically.We performed a detailed clinical and molecular characterization of five patients with deletions involving YWHAE but not PAFAH1B1, two with deletion including PAFAH1B1 but not YWHAE, and one with deletion of YWHAE and mosaic for deletion of PAFAH1B1.Three deletions were terminal whereas five were interstitial. Patients with deletions including YWHAE but not PAFAH1B1 presented with significant growth restriction, cognitive impairment, shared craniofacial features, and variable structural abnormalities of the brain. Growth restriction was not observed in one patient with deletion of YWHAE and TUSC5, implying that other genes in the region may have a role in regulation of growth with CRK being the most likely candidate. Using array based comparative genomic hybridisation and long range polymerase chain reaction, we have delineated the breakpoints of these nonrecurrent deletions and show that the interstitial genomic rearrangements are likely generated by diverse mechanisms, including the recently described Fork Stalling and Template Switching (FoSTeS)/Microhomology Mediated Break Induced Replication (MMBIR).Microdeletions of chromosome 17p13.3 involving YWHAE present with growth restriction, craniofacial dysmorphisms, structural abnormalities of brain and cognitive impairment. The interstitial deletions are mediated by diverse molecular mechanisms.

Background: Wolff-Parkinson-White syndrome (WPW) is a bypass reentrant tachycardia that results from an abnormal connection between the atria and ventricles. Mutations in PRKAG2 have been described in patients with familial WPW syndrome and hypertrophic cardiomyopathy. Based on the role of bone morphogenetic protein (BMP) signaling in the development of annulus fibrosus in mice, it has been proposed that BMP signaling through the type 1a receptor and other downstream components may play a role in preexcitation. Methods and Results: Using the array comparative genomic hybridization (CGH), we identified five individuals with non-recurrent deletions of 20p12.3. Four of these individuals had WPW syndrome with variable dysmorphisms and neurocognitive delay. With the exception of one maternally inherited deletion, all occurred de novo, and the smallest of these, harbored a single gene, BMP2. In two individuals with additional features of Alagille syndrome, deletion of both JAG1 and BMP2 were identified. Deletion of this region has not been described as a copy-number variant in the Database of Genomic Variants and has not been identified in 13,321 individuals from other cohort examined by array CGH in our laboratory. Conclusions: Our findings demonstrate a novel genomic disorder characterized by deletion of BMP2 with variable cognitive deficits and dysmorphic features and show that individuals bearing microdeletions in 20p12.3 often present with WPW syndrome.

Mitochondrial neurogastrointestinal encephalomyopathy (MNGIE) is an autosomal recessive multisystem disorder caused by thymidine phosphorylase (TP) deficiency, resulting in severe gastrointestinal dysmotility and skeletal muscle abnormalities. A patient is reported with a classical MNGIE clinical presentation but without skeletal muscle involvement at morphological, enzymatic, or mitochondrial DNA level, though gastrointestinal myopathy was present. MNGIE was diagnosed by markedly raised plasma thymidine and reduced thymidine phosphorylase activity. Molecular genetic analysis showed a homozygous novel splice site mutation in TP. On immunohistochemical studies there was marked TP expression in the CNS, in contrast to what has been observed in rodents. It is important to examine the most significantly affected tissue and to measure TP activity and plasma thymidine in order to arrive at an accurate diagnosis in this condition.

Jumping translocations (JTs) are very rare chromosome aberrations, usually identified in tumors. We report a constitutional JT between donor chromosome 21q21.3-->qter and recipients 13qter and 18qter, resulting in an approximately 15.5-Mb proximal deletion 21q in a girl with mild developmental delay and minor dysmorphic features. Using fluorescence in situ hybridization (FISH) studies, we identified an approximately 550-kb complex inter- and intra-chromosomal low-copy repeat (LCR) adjacent to the 21q21.3 translocation breakpoint. On the recipient chromosomes 13qter and 18qter, the telomeric sequences TTAGGG were retained. Genotyping revealed that the deletion was of maternal origin. We propose that genome architecture involving LCRs may be a major mechanism responsible for the origin of jumping translocations.

We report a 5-year-old boy with a small de novo marker chromosome derived from the proximal short arm of chromosome 17. His clinical features include hypotonia, global developmental delay, oval face with large nose and prominent ears, and ligamentous laxity of the fingers. Magnetic resonance imaging of the brain demonstrated mildly delayed myelination. G-band chromosome analysis revealed mosaicism for a small marker chromosome in 85% of the peripheral blood cells analyzed. Fluorescence in situ hybridization and microsatellite polymorphism studies showed that the der(17) was of maternal origin and included genetic material from the 17p10-p12 region, but did not contain the PMP22 gene. One breakpoint mapped within the centromere and the second breakpoint mapped adjacent to the Charcot-Marie-Tooth disease type 1A proximal low-copy repeat (CMT1A-REP). We compare the clinical characteristics of our patient with those previously reported to have a duplication involving the proximal short arm region of chromosome 17 to further delineate the phenotype of trisomy 17p10-p12.

Williams-Beuren syndrome (WBS) is a multisystemic genomic disorder typically caused by a recurrent ˜1.5-1.8 Mb deletion on 7q11.23. Atypical deletions can provide important insight into the genotype-phenotype correlations. Here, we report the phenotypic and molecular characterization of a girl with a de novo 81.8 kb deletion in the WBS critical region, which involves the ELN and LIMK1 genes only. The patient presented at 2 months of age with extensive vascular abnormalities, mild facial dysmorphism and delays in her fine motor skills. We discuss potential molecular mechanisms and the role of ELN and LIMK1 in the different phenotypic features. We compare the findings in our patient with previously reported overlapping deletions. The phenotypic variability among these patients suggests that other factors are important in the phenotype and possibly include: position effects related to copy number variation size, variations in the non-deleted alleles, genetic modifiers elsewhere in the genome, or reduced penetrance for specific phenotypes.

Repetitive element PCR (rep-PCR) uses outward-facing primers to amplify multiple segments of DNA located between conserved repeated sequences interspersed along the bacterial chromosome. Polymorphisms of rep-PCR amplification products can serve as strain-specific molecular fingerprints. Primers directed at the repetitive extragenic palindromic element were used to characterize isolates of Legionella pneumophila and other Legionella species. Substantial variation was seen among the rep-PCR fingerprints of different Legionella species and serogroups. More limited, but distinct, polymorphisms of the rep-PCR fingerprint were evident among epidemiologically unrelated isolates of L. pneumophila serogroup 1. Previously characterized Legionella isolates from nosocomial outbreaks were correctly clustered by this method. These results suggest the presence of repetitive extragenic palindromic-like elements within the genomes of members of the family Legionellaceae that can be used to discriminate between strains within a serogroup of L. pneumophila and between different Legionella species. rep-PCR appears to be a useful technique for the molecular fingerprinting of Legionella species.

Mutations affecting the peripheral myelin protein-22 (PMP22) gene have been shown to be associated with inherited peripheral neuropathies. To provide the molecular basis for the analysis of such mutations, we have cloned and characterized the human PMP22 gene. It spans approximately 40 kilobases and contains four coding exons. Detailed analysis of its 5‘-flanking region suggested the presence of two alternatively transcribed, but untranslated exons. Mapping of separate PMP22 mRNA transcription initiation sites to each of these exons indicates that PMP22 expression is regulated by two alternatively used promoters. In support of this hypothesis, both putative promoter sequences demonstrated the ability to drive expression of reporter genes in transfection experiments. Furthermore, the structures of the 5‘-portions of the PMP22 genes appear to be identical in rat and human, supporting the biological significance of the observed arrangement of regulatory regions. The relative expression of the alternative PMP22 transcripts is tissue-specific, and high levels of the exon 1A-containing transcript are tightly coupled to myelin formation. In contrast, exon 1B-containing transcripts are predominant in non-neural tissues and in growth-arrested primary fibroblasts. Interestingly, although a strong upregulation of PMP22 mRNA was observed in cultured Schwann cells in the presence of the adenylate cyclase activator forskolin under various culture conditions, the regulation of the different PMP22 mRNA species did not mimic the regulation that occurs during myelin formation in vivo. The observed regulation of the PMP22 gene by a complex molecular mechanism is consistent with the proposed dual role of PMP22 in neural and non-neural tissue.

Objectives: Copy number variants (CNVs) have been recognized as a source of genetic variation that contributes to disease phenotypes. Alzheimer disease (AD) has high heritability for occurrence and age at onset (AAO). We performed a cases-only genome-wide CNV association study for age at onset of AD. Methods: The discovery case series (n = 40 subjects with AD) was evaluated using array comparative genome hybridization (aCGH). A replication case series (n = 507 subjects with AD) was evaluated using Affymetrix array (n = 243) and multiplex ligation-dependent probe amplification (n = 264). Hazard models related onset age to CNV. Results: The discovery sample identified a chromosomal segment on 14q11.2 (19.3–19.5 Mb, NCBI build 36, UCSC hg18 March 2006) as a region of interest (genome-wide adjusted p = 0.032) for association with AAO of AD. This region encompasses a cluster of olfactory receptors. The replication sample confirmed the association (p = 0.035). The association was found for each APOE4 gene dosage (0, 1, and 2). Conclusion: High copy number in the olfactory receptor region on 14q11.2 is associated with younger age at onset of AD.

Background: Microdeletions within chromosome 15q13.3 are associated both with a recently recognised syndrome of mental retardation, seizures, and dysmorphic features, and with schizophrenia. Methods and results: Based on routine diagnostic testing of ∼8200 samples using array comparative genomic hybridisation, we identified 20 individuals (14 children and six parents in 12 families) with microdeletions of 15q13.3. Phenotypes in the children included developmental delay, mental retardation, or borderline IQ in most and autistic spectrum disorder (6/14), speech delay, aggressiveness, attention deficit hyperactivity disorder, and other behavioural problems. Both parents were available in seven families, and the deletion was de novo in one, inherited from an apparently normal parent in four, and inherited from a parent with learning disability and bipolar disorder in two families. Of the 14 children, six in five families were adopted, and DNA was available for only one of these 10 biological parents; the deletion was very likely inherited for one of these families with two affected children. Among the unavailable parents, two mothers were described as having mental retardation, another mother as having “mental illness”, and one father as having schizophrenia. We hypothesise that some of the unavailable parents have the deletion. Conclusions: The occurrence of increased adoption, frequent autism, bipolar disorder, and lack of penetrance are noteworthy findings in individuals with deletion 15q13.3. A high rate of adoption may be related to the presence of the deletion in biological parents. Unconfirmed histories of antisocial behaviours in unavailable biological parents raise the concern that future research may show that deletion 15q13.3 is associated with such behaviours.

Contains fulltext : 80905.pdf (Publisher’s version ) (Closed access) Mutations in ROR2, encoding a receptor tyrosine kinase, can cause autosomal recessive Robinow syndrome (RRS), a severe skeletal dysplasia with limb shortening, brachydactyly, and a dysmorphic facial appearance. Other mutations in ROR2 result in the autosomal dominant disease, brachydactyly type B (BDB1). No functional mechanisms have been delineated to effectively explain the association between mutations and different modes of inheritance causing different phenotypes. BDB1-causing mutations in ROR2 result from heterozygous premature termination codons (PTCs) in downstream exons and the conveyed phenotype segregates as an autosomal dominant trait, whereas heterozygous missense mutations and PTCs in upstream exons result in carrier status for RRS. Given that the distribution of PTC mutations revealed a correlation between the phenotype and the mode of inheritance conveyed, we investigated the potential role for the nonsense-mediated decay (NMD) pathway in the abrogation of possible aberrant effects of selected mutant alleles. Our experiments show that triggering or escaping NMD may cause different phenotypes with a distinct mode of inheritance. We generalize these findings to other disease-associated genes by examining PTC mutation distribution correlation with conveyed phenotype and inheritance patterns. Indeed, NMD may explain distinct phenotypes and different inheritance patterns conveyed by allelic truncating mutations enabling better genotype-phenotype correlations in several other disorders.

Cornelia de Lange syndrome (CdLS) is a multisystem congenital anomaly disorder. Heterozygous point mutations in three genes (NIPBL, SMC3 and SMC1A), encoding components of the sister chromatid cohesion apparatus, are responsible for approximately 50-60% of CdLS cases. Recent studies have revealed a high degree of genomic rearrangements (for example, deletions and duplications) in the human genome, which result in gene copy number variations (CNVs). CNVs have been associated with a wide range of both Mendelian and complex traits including disease phenotypes such as Charcot-Marie-Tooth type 1A, Pelizaeus-Merzbacher, Parkinson, Alzheimer, autism and schizophrenia. Increased versus decreased copy number of the same gene can potentially cause either similar or different clinical features.This study identified duplications on chromosomes 5 or X using genome wide array comparative genomic hybridisation (aCGH). The duplicated regions contain either the NIPBL or the SMC1A genes. Junction sequences analyses revealed the involvement of three genomic rearrangement mechanisms. The patients share some common features including mental retardation, developmental delay, sleep abnormalities, and craniofacial and limb defects. The systems affected are the same as in CdLS, but clinical manifestations are distinct from CdLS; particularly the absence of the CdLS facial gestalt.The results confirm the notion that duplication CNV of genes can be a common mechanism for human genetic diseases. Defining the clinical consequences for a specific gene dosage alteration represents a new "reverse genomics" trend in medical genetics that is reciprocal to the traditional approach of delineation of the common clinical phenotype preceding the discovery of the genetic aetiology.

In the past fifteen years, an emerging group of genetic diseases have been described that result from DNA rearrangements rather than from single nucleotide changes. Such conditions have been referred to as genomic disorders. The predominant molecular mechanism underlying the rearrangements that cause this group of diseases and traits is nonallelic homologous recombination (NAHR) (unequal crossing-over between chromatids or chromosomes) utilizing low-copy repeats (LCRs) (also known as segmental duplications) as substrates. In contradistinction to highly repetitive sequences (e.g. Alu and LINE elements), these higher-order genomic architectural features usually span1kb and up to hundreds of kilobases of genomic DNA, share96% sequence identity and constitute5% of the human genome. Many LCRs have complex structure and have arisen during primate speciation as a result of serial segmental duplications. LCRs can stimulate and/or mediate constitutional (both recurrent and nonrecurrent), evolutionary, and somatic rearrangements. Recently, copy-number variations (CNVs), also referred to as large-scale copy-number variations (LCVs) or copy-number polymorphisms (CNPs), parenthetically often associated with LCRs, have been demonstrated as a source of human variation as well as a potential cause of diseases. In addition to fluorescence in situ hybridization (FISH), pulsed-field gel electrophoresis (PFGE), and in silico analyses, multiplex ligation-dependent probe amplification (MLPA), and array comparative genomic hybridization (aCGH) with BAC and PAC clones have proven to be useful diagnostic methods for the detection and characterization of DNA rearrangements with the latter enabling high-resolution genome-wide analysis. The clinical implementation of such techniques is revolutionizing clinical cytogenetics.

This report describes the cloning and sequencing of a chromosomally encoded tetracycline resistance determinant from a clinical isolate of methicillin-resistant Staphylococcus aureus. On the basis of the sequence, the gene is in the tet(M) class, and it was shown that the S. aureus tetA(M) gene is induced at the level of transcription.

We present molecular genetic investigations of a 4-year-old boy with craniofacial dysmorphism and developmental delay. Trivial mitral and tricuspid regurgitation without gross structural abnormality was diagnosed by echocardiography. High-resolution chromosome analysis revealed an interstitial deletion, del(10)(p12.1p12.32). To characterize the deletion size and breakpoints, we performed fluorescence in situ hybridization analysis using 27 BAC clones. Our data demonstrate an approximately 5.5 Mb deletion del(10)(p12.1p12.31). Surprisingly, the BAC clone RP11-56H7 that contains NEBL, an apparent downstream gene of the cardiogenic transcription factor HAND2 previously shown to be deleted in the patients with DiGeorge 2 syndrome and 10p13 deletion, was deleted in our patient with 10p12.1-p12.31 deletion. In addition, we provide clinical data and results of molecular analysis for a patient with multiple congenital anomalies including Ebstein's anomaly, kidney malformations, and 10p13-p14 deletion. We also reviewed 19 patients with congenital heart defects and deletions involving 10p and propose that atrial septal defect (ASD) is a common cardiac anomaly associated with DiGeorge 2 syndrome. Based on genotype-phenotype analysis of published patients and those reported herein, we propose an approximately 1.0 Mb critical region between loci D10S547 and D10S2176 in 10p14 to be associated with ASD. Considering that septal defects are the most frequent congenital heart anomaly, we suggest that further investigations in the 10p critical region are important to identify gene(s) responsible for this common birth defect.

Background: Proximal chromosome 17p is a region rich in low copy repeats (LCRs) and prone to chromosomal rearrangements. Four genomic disorders map within the interval 17p11–p12: Charcot–Marie–Tooth disease type 1A, hereditary neuropathy with liability to pressure palsies, Smith–Magenis syndrome, and dup(17)(p11.2p11.2) syndrome. While 80–90% or more of the rearrangements resulting in each disorder are recurrent, several non-recurrent deletions or duplications of varying sizes within proximal 17p also have been characterised using fluorescence in situ hybridisation (FISH). Methods: A BAC/PAC array based comparative genomic hybridisation (array-CGH) method was tested for its ability to detect these genomic dosage differences and map breakpoints in 25 patients with recurrent and non-recurrent rearrangements. Results: Array-CGH detected the dosage imbalances resulting from either deletion or duplication in all the samples examined. The array-CGH approach, in combination with a dependent statistical inference method, mapped 45/46 (97.8%) of the analysed breakpoints to within one overlapping BAC/PAC clone, compared with determinations done independently by FISH. Several clones within the array that contained large LCRs did not have an adverse effect on the interpretation of the array-CGH data. Conclusions: Array-CGH is an accurate and sensitive method for detecting genomic dosage differences and identifying rearrangement breakpoints, even in LCR-rich regions of the genome.

We report a 5-year-old boy with a small de novo marker chromosome derived from the proximal short arm of chromosome 17. His clinical features include hypotonia, global developmental delay, oval face with large nose and prominent ears, and ligamentous laxity of the fingers. Magnetic resonance imaging of the brain demonstrated mildly delayed myelination. G-band chromosome analysis revealed mosaicism for a small marker chromosome in 85% of the peripheral blood cells analyzed. Fluorescence in situ hybridization and microsatellite polymorphism studies showed that the der(17) was of maternal origin and included genetic material from the 17p10-p12 region, but did not contain the PMP22 gene. One breakpoint mapped within the centromere and the second breakpoint mapped adjacent to the Charcot-Marie-Tooth disease type 1A proximal low-copy repeat (CMT1A-REP). We compare the clinical characteristics of our patient with those previously reported to have a duplication involving the proximal short arm region of chromosome 17 to further delineate the phenotype of trisomy 17pl0-p12.

Fundus albipunctatus (FA; OMIM 136880) is a rare form of apparently stationary night blindness characterized by the presence of myriad symmetrical round white dots in the fundus with a greater concentration in the midperiphery. A distantly similar but distinct clinical entity, retinitis punctata albescens (RPA), is also characterized by aggregation of irregular white flecks but is progressive and evolves to generalized atrophy of the retina. We studied 4 consanguineous kindreds diagnosed with FA from Saudi Arabia. Given the substantial phenotypic variation and overlap between different flecked retinal dystrophies, we evaluated all known genes associated with such conditions by both genetic analysis and direct sequencing. In one kindred, KKESH-099, we identified a homozygous R150Q alteration in RLBP1, the gene encoding the cellular retinaldehyde binding protein, associated previously with both recessive retinitis pigmentosa (arRP) and RPA. Examination of several patients aged 3-20 years over a 9-year period presented no evidence for either RP or RPA. In contrast, clinical examination of individuals with the same mutation in their fourth and fifth decade revealed signs consistent with RPA. The data suggest that the R150Q mutation in RLBP1 may result in RPA with slow progression. More importantly, younger individuals diagnosed with the milder disorder FA thought to be stationary may evolve to a more devastating and progressive phenotype.

Discovery of the gene responsible for a recessive, early-onset maculopathy has focused attention on a biomolecular pathway in which subtle metabolic flaws become toxic to irreplaceable retinal cells. The pathway's identification points toward specific ways of intervening. Among patients with age-related macular degeneration, carriers of single mutations in the same gene are proving to be remarkably common.

X-linked Charcot-Marie-Tooth disease (CMTX) is the second most common form of Charcot-Marie-Tooth disease. Variable histopathological and nerve conduction velocity (NCV) results have suggested either a primary demyelinating or axonal polyneuropathy. We identified five individuals across three generations in a family with CMTX associated with a mutation in the gene coding for connexin 32. All individuals were studied by clinical neurological examination, DNA analysis, and nerve conduction studies. The proband (1174/KD) also underwent a sural nerve biopsy. As expected, all the affected males were more clinically affected than the females. All affected males and obligate female carriers exhibited some electrophysiological characteristics of demyelination. However, striking heterogeneity of nerve conduction velocities was seen. This family shows that CMTX is a heterogeneous and distinctly nonuniform demyelinating polyneuropathy, the severity of which varies with sex and age. Such electrophysiological variability is unique among hereditary neuropathies.

Charcot-Marie-Tooth disease (CMT) is the most common cause of peripheral neuropathy, with an incidence of 1: 2500 persons affected. CMT1A is caused by a submicroscopic duplication in 17p12. Several methods exist for determining a diagnosis in an individual. Many of these methods are not suitable for prenatal diagnosis. Previously, we reported the use of fluorescence in situ hybridization (FISH) to detect the common duplication found in more than 98% of individuals with CMT1A. We also have reported the validation of the FISH assay for amniotic fluid specimens and chorionic villus samples. Herein, we report our experience with testing for CMT1A in prenatal specimens.

We describe an unique patient presenting with severe leukodystrophy compatible with Pelizaeus-Merzbacher disease and peripheral neuropathy consistent with Charcot-Marie-Tooth disease type 1 in addition to Waardenburg-Hirschsprung syndrome. A novel mutation was identified in her SOX10 gene, which encodes a transcription factor preferentially expressed in the late embryonic glial cell lineage and in mature myelin-forming cells of both the central nervous system and peripheral nervous system, as well as in the early neural crest cells. Heterozygous SOX10 loss-of-function mutations have been reported in patients with Waardenburg-Hirschsprung syndrome and its murine model, Dominant megacolon. However, neither Waardenburg-Hirschsprung syndrome patients nor Dominant megacolon mice have dysmyelinating features, suggesting the question of how SOX10 acts in the glial lineage in vivo. The novel mutation described herein does not disrupt the coding region but extends the peptide and hence is likely to act as a dominant-negative allele. Our findings indicate that dysfunction of SOX10 may lead to deficiency of myelination in the central nervous system and peripheral nervous system as well as hypopigmentation and enteric aganglionosis.

Charcot-Marie-Tooth Disease (CMT) is the most common cause of peripheral neuropathy, with an incidence of 1:2500 persons affected. Previously, we reported the use of fluorescence in situ hybridization (FISH) to detect the common submicroscopic duplication of 17p12 found in more than 98 per cent of individuals with CMT1A. We found that FISH is a reliable means for the diagnosis of the duplication of 17p12 in peripheral blood and reported the validation of the FISH assay for amniotic fluid specimens. Herein, we report the validation of the FISH assay for use on chorionic villus samples (CVS) to prenatally diagnose CMT1A duplications and the testing of 17 prenatal specimens. Seven fetuses were found to carry the duplication and are predicted to be affected. FISH is a rapid assay in prenatal specimens, with a 9.3 day average turn-around time. Limited follow-up on pregnancies indicates that the duplication found in CMT1A is reliably diagnosed in the fetus, using FISH on either amniotic fluid specimens or CVS.

Williams syndrome (WS) is a neurodevelopmental disorder with a variable phenotype. Molecular genetic studies have indicated that hemizygosity at the elastin locus (ELN) may account for the cardiac abnormalities seen in WS, but that mental retardation and hypercalcemia are likely caused by other genes flanking ELN. In this study, we defined the minimal critical deletion region in 63 patients using 10 microsatellite markers and 5 fluorescence in situ hybridization (FISH) probes on chromosome 7q, flanking ELN. The haplotype analyses showed the deleted cases to have deletions of consistent size, as did the FISH analyses using genomic probes for the known ends of the commonly deleted region defined by the satellite markers. In all informative cases deleted at ELN, the deletion extends from D7S489U to D7S1870. The genetic distance between these two markers is about 2 cM. Of the 51 informative patients with deletions, 29 were maternal and 22 were paternal in origin. There was no evidence for effects on stature by examining gender, ethnicity, cardiac status, or parental origin of the deletion. Heteroduplex analysis for LIMK1, a candidate gene previously implicated in the WS phenotype, did not show any mutations in our WS patients not deleted for ELN. LIMK1 deletions were found in all elastin-deletion cases who had WS. One case, who has isolated, supravalvular aortic stenosis and an elastin deletion, was not deleted for LIMK1. It remains to be determined if haploinsufficiency of LIMK1 is responsible in part for the WS phenotype or is simply deleted due to its close proximity to the elastin locus.

We report on a newborn girl with malformed ears, bilateral cleft lip and cleft palate, complex congenital heart disease, absent left thumb, and rib abnormalities. Cytogenetic analysis demonstrated a de novo interstitial deletion of the short arm of chromosome 1 [46,XX,del(1)(p21p22.3)]. Reports of interstitial deletions on the short arm of chromosome 1 are rare. However, when comparing this patient's phenotype to others with deletions of 1p, we found that the current case was much more severely affected than previously reported cases.

Charcot-Marie-Tooth (CMT) disease type 1A is an inherited peripheral neuropathy characterized by slowly progressive distal muscle wasting and weakness, decreased nerve conduction velocities, and genetic linkage to 17p12. Most (98%) CMT1A cases are caused by a DNA duplication of a 1.5-Mb region in 17p12 containing the PMP22 gene. The reciprocal product of the CMT1A duplication is a 1.5-Mb deletion which causes hereditary neuropathy with liability to pressure palsies (HNPP). The most informative current diagnostic testing requires pulsed-field gel electrophoresis to detect DNA rearrangement-specific junction fragments. We investigated the use of interphase FISH for the detection of duplications and deletions for these disorders in the clinical molecular cytogenetics laboratory. Established cell lines or blood specimens from 23 individuals with known molecular diagnoses and 10 controls were obtained and scored using a two-color FISH assay. At least 70% of CMT1A cells displayed three signals consistent with duplications. Using this minimum expected percentile to make a CMT1A duplication diagnosis, all patients with CMT1A showed a range of 71-92% of cells displaying at least three signals. Of the HNPP cases, 88% of cells displayed only one hybridization signal, consistent with deletions. The PMP22 locus from normal control individuals displayed a duplication pattern in approximately 9% of cells, interpreted as replication of this locus. The percentage of cells showing replication was significantly lower than in those cells displaying true duplications. We conclude that FISH can be reliably used to diagnose CMT1A and HNPP in the clinical cytogenetics laboratory and to readily distinguish the DNA rearrangements associated with these disorders from individuals without duplication or deletion of the PMP22 locus.

Dejerine-Sottas syndrome (DSS), a severe demyelinating peripheral neuropathy with onset in infancy, has been associated with mutations in either PMP22 or MPZ. Most cases of DSS are caused by a single heterozygous dominant point mutation. We identified three de novo point mutations in MPZ exon 3 in a sporadic DSS patient. These three point mutations occur on the same allele and result in three novel amino acid substitutions: Ile(85)Thr, Asn(87)His, and Asp(99)Asn. Our data raise the question as to the potential mechanism(s) involved in the formation of multiple point mutations at a given locus.

Primary congenital glaucoma is an uncommon autosomal recessive condition that results from a developmental defect in the trabecular meshwork and anterior chamber angle, manifesting in the neonatal or infantile period with increased intraocular pressure, corneal enlargement and edema, and optic nerve cupping with consequent loss of vision. Nothing is known about its genetic location.Linkage analysis was performed in 25 primary congenital glaucoma Saudi Arabian families with six polymorphic DNA markers on chromosome 1q in a region that has shown tight linkage to a locus for autosomal dominant juvenile-onset open angle glaucoma (GLC1A). Twenty-four of these families are highly consanguineous.Each family was shown separately to exclude the 8-centimorgan (cM) interval containing the GLC1A locus. Four families independently demonstrated overlapping regions of exclusion (thetaor = -2) that spanned the entire 8-cM interval. Assignment of a primary congenital glaucoma locus in this region could be excluded by a cadre of 21 families because a primary congenital glaucoma disease locus did not segregate in an autosomal recessive manner on haplotypes constructed with markers in this region. For all families, no affected individuals demonstrated homozygosity of alleles in regions tightly linked to the GLC1A locus.These results exclude the 8-cM region on chromosome 1q shown to contain the GLC1A locus from containing a disease locus for primary congenital glaucoma in this population of 25 Saudi Arabian families.

Autosomal dominant Charcot-Marie-Tooth type-1A neuropathy (CMT1A) is a demyelinating peripheral nerve disorder that is commonly associated with a submicroscopic tandem DNA duplication of a 1.5-Mb region of 17p11.2p12 that contains the peripheral myelin gene PMP22. Clinical features of CMT1A include progressive distal muscle atrophy and weakness, foot and hand deformities, gait abnormalities, absent reflexes, and the completely penetrant electrophysiologic phenotype of symmetric reductions in motor nerve conduction velocities (NCVs). Molecular and fluorescense in situ hybridization (FISH) analyses were performed to determine the duplication status of the PMP22 gene in four patients with rare cytogenetic duplications of 17p. Neuropathologic features of CMT1A were seen in two of these four patients, in addition to the complex phenotype asociated with 17p partial trisomy. Our findings show that the CMT1A phenotype of reduced NCV is specifically associated with PMP22 gene duplications, thus providing further support for the PMP22 gene dosage mechanism for CMT1A.

Smith-Magenis syndrome (SMS) is a multiple congenital anomaly, mental retardation (MCA/MR) syndrome associated with deletion of chromosome 17 band p11.2. As part of a multi-disciplinary clinical, cytogenetic, and molecular approach to SMS, detailed clinical studies including radiographic, neurologic, developmental, ophthalmologic, otolaryngologic, and audiologic evaluations were performed on 27 SMS patients. Significant findings include otolaryngologic abnormalities in 94%, eye abnormalities in 85%, sleep abnormalities (especially reduced REM sleep) in 75%, hearing impairment in 68% (approximately 65% conductive and 35% sensorineural), scoliosis in 65%, brain abnormalities (predominantly ventriculomegaly) in 52%, cardiac abnormalities in at least 37%, renal anomalies (especially duplication of the collecting system) in 35%, low thyroxine levels in 29%, low immunoglobulin levels in 23%, and forearm abnormalities in 16%. The measured IQ ranged between 20-78, most patients falling in the moderate range of mental retardation at 40-54, although several patients scored in the mild or borderline range. The frequency of these many abnormalities in SMS suggests that patients should be evaluated thoroughly for associated complications both at the time of diagnosis and at least annually thereafter.

This study presents a longitudinal comparison of motor nerve conduction velocities (MCVs) in patients with Charcot-Marie-Tooth type 1A with proven duplication of a segment of chromosome 17p11.2p12. Results were compared for 8 CMT1A duplication patients from one family whose MCV measurements were taken 22 years apart (1967 and 1989). Measurements from a total of seven median motor and five peroneal motor MCVs were compared. Median MCVs showed a slight reduction that averaged 2.2 m/s, and peroneal MCVs showed an average decrease of 3.0 m/s. In addition, mild objective increase in limb weakness was seen in only 1 of 8 patients and subjective symptoms of gradual worsening of leg strength were noted in half the patients over the same period. In this study of a small group of CMT1A patients with proven segmental duplication of chromosome 17p11.2p12, the motor conduction velocities and clinical motor exam did not change significantly over 22 years.

The myelin protein zero gene (MPZ) maps to chromosome 1q22-q23 and encodes the most abundant peripheral nerve myelin protein. The Po protein functions as a homophilic adhesion molecule in myelin compaction. Mutations in the MPZ gene are associated with the demyelinating peripheral neuropathies Charcot-Marie-Tooth disease type 1B (CMT1B), and the more severe Dejerine-Sottas syndrome (DSS). We have surveyed a cohort of 70 unrelated patients with demyelinating polyneuropathy for additional mutations in the MPZ gene. The 1.5-Mb DNA duplication on chromosome 17p11.2-p12 associated with CMT type 1A (CMT1A) was not present. By DNA heteroduplex analysis, four base mismatches were detected in three exons of MPZ. Nucleotide sequence analysis identified a de novo mutation in MPZ exon 3 that predicts an Ile(135)Thr substitution in a family with clinically severe early-onset CMT1, and an exon 3 mutation encoding a Gly(137)Ser substitution was identified in a second CMT1 family. Each predicted amino acid substitution resides in the extracellular domain of the Po protein. Heteroduplex analysis did not detect either base change in 104 unrelated controls, indicating that these substitutions are disease-associated mutations rather than common polymorphisms. In addition, two polymorphic mutations were identified in MPZ exon 5 and exon 6, which do not alter the codons for Gly(200) and Ser(228), respectively. These observations provide further confirmation of the role of MPZ in CMT1B and suggest that MPZ coding region mutations may account for a limited percentage of disease-causing mutations in nonduplication CMT1 patients.

Methicillin resistance in Staphylococcus aureus is a frequent cause of nosocomial and community-acquired infections. Accurate, rapid epidemiologic typing is crucial to the identification of the source and spread of infectious disease and could provide detailed information on the generation of methicillin-resistant S. aureus (MRSA) strains. The high degree of genetic relatedness of MRSA strains has precluded the use of more conventional methods of genetic fingerprinting. A rapid DNA fingerprinting method that exploits PCR amplification from a DNA repeat sequence in MRSA is described. The random chromosomal distribution of this repeat sequence provides an ideal target for detecting DNA fragment patterns specific to individual MRSA strains. Two PCR fingerprinting methods which use an oligonucleotide primer based on a repetitive sequence found in Mycoplasma pneumoniae are presented. The repetitive element sequence-based PCR (rep-PCR) and fluorophore-enhanced rep-PCR (FERP) can identify epidemic strains among background MRSA. The combination of oligonucleotide primers labeled with different fluorescent dyes allowed simultaneous FERP fingerprinting and mecA gene detection. Eight different fingerprint patterns were observed in MRSA strains collected from different sources. These techniques provide a rapid discriminatory means of molecular epidemiologic typing of MRSA involved in nosocomial infections.

Fluorophore-labeled oligonucleotide primers complementary to defined interspersed repetitive sequences conserved in diverse bacteria were used in the polymerase chain reaction to generate DNA fingerprint patterns from selected pathogenic bacteria. Fluorophore-enhanced, repetitive sequence-based polymerase chain reaction allowed discrimination between unrelated isolates of penicillin-resistant Streptococcus pneumoniae recovered from pediatric patients and Mycobacterium avium cultured from patients with acquired immunodeficiency syndrome. Combinations of oligonucleotide primers labeled with distinct fluorescent dyes enabled simultaneous DNA fingerprinting and Shiga-like toxin gene detection in enterohemorrhagic Escherichia coli isolates. Fluorophore-enhanced, repetitive sequence-based polymerase chain reaction was performed with either purified DNA or intact cells that were lysed during the polymerase chain reaction. Fluorophore-enhanced, repetitive sequence-based polymerase chain reaction successfully combines polymerase chain reaction amplification and fluorescent label detection for DNA fingerprinting of cultured bacterial pathogens.

Collectively, the inherited disorders of peripheral nerves represent a common group of neurological diseases and are frequently encountered in the clinical setting. Recent advances in molecular genetics have not only provided improved diagnosis and counselling, but may ultimately lead to specific, rational therapies for the various forms of inherited neuropathy. Charcot-Marie-Tooth neuropathy type 1 (CMT1) is a genetically heterogeneous group of chronic demyelinating polyneuropathies with loci mapping to chromosome 17p (CMT1A), chromosome 1q (CMT1B), the X chromosome (CMTX) and to another unknown autosome (CMT1C). CMT1A is most often associated with a tandem 1.5-megabase (Mb) duplication in chromosome 17p11.2-12, or may occasionally result from a point mutation in the peripheral myelin protein 22 (PMP22) gene. CMT1B is associated with point mutations in the myelin protein zero (P0) gene. The molecular defect in CMT1C is unknown. CMTX is associated with defects in the connexin 32 gene. Charcot-Marie-Tooth neuropathy type 2 (CMT2) is an axonal neuropathy, also of undetermined cause. One locus for CMT2 has been assigned for chromosome 1p (CMT2A). Dejerine-Sottas disease is a severe, infantile-onset, demyelinating polyneuropathy which may be associated with point mutations in the P0 or PMP22 genes. Hereditary neuropathy with liability to pressure palsies (HNPP) is an autosomal dominant disorder that results in a recurrent, episodic demyelinating neuropathy. HNPP is associated with a 1.5-Mb deletion in chromosome 17p11.2-12 and may result from reduced expression of the PMP22 gene. CMT1A and HNPP are apparent reciprocal duplication/deletion syndromes originating from unequal cross-over during germ-cell meiosis.