Molecular analysis of the induction of immunoglobulin E synthesis in human B cells by interleukin 4 and engagement of CD40 antigen.

The molecular events leading to immunoglobulin E (IgE) synthesis in human sIgE- B cells stimulated with interleukin 4 (IL-4) and anti-CD40 monoclonal antibody (mAb) 626.1 were analyzed. Anti-CD40 mAb increased the levels of IL-4-induced germline C epsilon transcripts and induced the production of mature C epsilon mRNA. These effects were dependent on the presence of IL-4. Nested primer PCR revealed deletional switch recombination occurring only in B cell stimulated with both IL-4 and anti-CD40 mAb. DNA sequence analysis of switch fragments showed direct S mu/S epsilon joining, without the deletions or duplications within S mu often found in B cells stimulated with IL-4 and Epstein-Barr virus. Analysis of the switch junction map sites showed "hot spots" for recombination within S mu, but not within S epsilon. These findings indicate that IL-4 provides a signal to B cells to induce germline C epsilon transcription and concurrent CD40 engagement induces S mu/S epsilon deletional switch recombination, production of mature C epsilon mRNA, and IgE synthesis.

Eight years experience with enzyme replacement therapy in two children and one adult with Pompe disease.

Pompe disease (type 2 glycogenosis, acid maltase deficiency) is a disorder affecting skeletal and cardiac muscle, caused by deficiency of acid alpha-glucosidase. In 2006 enzyme therapy with recombinant human alpha-glucosidase received marketing approval based on studies in infants. Results in older children and adults are awaited. Earlier we reported on the 3-year follow-up data of enzyme therapy in two adolescents and one adult. In the present study these patients were followed for another 5 years. Two severely affected patients, wheelchair and ventilator dependent, who had shown stabilization of pulmonary and muscle function in the first 3 years, maintained this stabilization over the 5-year extension period. In addition patients became more independent in daily life activities and quality of life improved. The third moderately affected patient had shown a remarkable improvement in muscle strength and regained the ability to walk over the first period. He showed further improvement of strength and reached normal values for age during the extension phase. The results indicate that both long-term follow-up and timing of treatment are important topics for future studies.

Primary Care Provider Management of Congenital Hypothyroidism Identified Through Newborn Screening.

OBJECTIVE: To assess Primary Congenital Hypothyroidism (CH) management patterns and feasibility of providing long-term care for patients with CH identified through newborn screening by Primary Care Providers (PCPs) in California and Hawaii. STUDY DESIGN: A survey was mailed to all physicians (N=823) listed as the referral doctor for confirmed patients with CH identified through newborn screening programs in both states between 01/01/2009-12/31/2013. Information was collected on CH management patterns, barriers to providing care, and knowledge on CH treatment. Descriptive statistics and bivariate logistic regression results were reported. RESULTS: 206 PCPs completed the survey. Among these, 78% currently have patients with CH and 91% indicated willingness to provide long-term care to new patients with CH. Among PCPs currently caring for patients with CH, 17% managed CH by themselves with limited assistance from endocrinologists; 63% were involved in managing CH but endocrinologists played a larger role than PCPs; 19% were not involved in CH care. Only 49% of PCPs correctly answered questions regarding recommended follow-up frequencies and 23% knew the correct age for a trial off levothyroxine for suspected transient CH. Top two perceived barriers to providing long-term care included "need guidance or support from endocrinologists" (61%) and "not familiar with CH treatment guidelines" (28%). CONCLUSION: The majority of PCPs surveyed are willing to provide long-term care to patients with CH, but need support from endocrinologists and increased knowledge about current treatment guidelines.

Hybrid protein thymidine kinase gene fusions: plasmid vectors for the study of transcription and translation initiation signals.

The thymidine kinase (TK) gene (tk) from Herpes simplex virus type 1 has been used to form gene fusions encoding enzymatically active hybrid proteins. The promoter, translation initiation region, and the first three codons of the tk gene were removed and replaced with a series of DNA restriction sites. DNA fragments containing gene initiation regions were cloned into these sites and shown to synthesize enzymatically active proteins in Escherichia coli. These gene fusions were shown to complement an E. coli strain which is deficient in TK function. Gene initiation regions were used from the lac operon, the tnpR gene of Tn3, and the insA gene of ISl. TK synthesis was regulated by the control signals of the promoter fused to tk, and was dependent upon the phase alignment of the codons at the fusion joint. The size of the resulting protein was shown to be increased over the size of the original TK protein by the length of the coding region fused to TK. This demonstrated that the tk gene has non-essential N-terminal amino acids that can be replaced by other amino acid sequences with the retention of TK enzymatic activity. Such tk gene fusions are useful in situations where fusions with other genes cannot be conveniently selected or assayed.

New versatile plasmid vectors for expression of hybrid proteins coded by a cloned gene fused to lacZ gene sequences encoding an enzymatically active carboxy-terminal portion of beta-galactosidase.

A new class of plasmid cloning vectors has been constructed with cleavage sites in a variety of translational reading phases of the promotorless lacZ gene. Fused hybrid proteins can be produced by these vectors by cloning DNA fragments containing the promoter, translation initiation site, and the amino terminal portion of a gene, all with proper orientation, into the correct translational reading frame of the lacZ gene. Enzymatically active hybrid-beta-galactosidase proteins are formed, which have amino-terminal amino acids encoded by the cloned gene segment. Another class of these vectors retains an active lac promoter and lacZ translation-initiation region, which can direct hybrid protein synthesis from DNA fragments that do not have gene initiation regions. These vectors allow transcription from the lacZ initiation region to proceed across, or to stop and restart within, an inserted fragment into the essential part of the beta-galactosidase gene. Also described is a small lacZ gene fragment (cartridge), without a plasmid replicon and without any other lac genes, which can be inserted directly into other genes to form hybrid protein fusions. Polyrestriction site sequences were easily moved into some of these vectors by incorporating drug-resistance genes that serve as markers for the selection and detection of these sequences; those markers can be easily removed afterwards.

Constitutional mosaicism for a chromosome 9 inversion resulting in recombinant aneusomy in an offspring.

We report on a case of constitutional mosaicism for a large pericentric inversion of chromosome 9 in a man whose daughter had recombinant aneusomy resulting in partial 9q duplication and partial 9p deletion. At age 6 months, the girl was evaluated because of congenital anomalies [corrected] and developmental delay. Chromosomal analysis on this infant showed a derivative chromosome 9 which was later determined to be a recombinant chromosome with trisomy of 9q34.1-->qter and monosomy of pter-->9p24. Chromosomal analysis in her father showed the presence of two cell lines; 75% of lymphocytes had a 46,XY pattern, and 25% had a 46,XY,inv(9)(p24q34.1) karyotype. The infant's physical findings represent a composite of the reported cases of both trisomy 9q34.1-->qter and monosomy pter-->9p24. The infant's father was phenotypically and cognitively normal. This case broadens the spectrum of reported cases of mosaicism for an autosomal structural rearrangement generating unbalanced gametes, and further supports the tenet that constitutional mosaicism has clinical relevance for genetic counseling.

Submicroscopic deletions at 22q11.2: variability of the clinical picture and delineation of a commonly deleted region.

DiGeorge anomaly (DGA) and velo-cardiofacial syndrome (VCFS) are frequently associated with monosomy of chromosome region 22q11. Most patients have a submicroscopic deletion, recently estimated to be at least 1-2 Mb. It is not clear whether individuals who present with only some of the features of these conditions have the deletion, and if so, whether the size of the deletion varies from those with more classic phenotypes. We have used fluorescence in situ hybridization (FISH) to assess the deletion status of 85 individuals referred to us for molecular analysis, with a wide range of DGA-like or VCFS-like clinical features. The test probe used was the cosmid sc11.1, which detects two loci about 2 Mb apart in 22q11.2. Twenty-four patients carried the deletion. Of the deleted patients, most had classic DGA or VCFS phenotypes, but 6 deleted patients had mild phenotypes, including 2 with minor facial anomalies and velopharyngeal incompetence as the only presenting signs. Despite the great phenotypic variability among the deleted patients, none had a deletion smaller than the 2-Mb region defined by sc11.1. Smaller deletions were not detected in patients with particularly suggestive phenotypes who were not deleted for sc11.1, even when tested with two other probes from the DGA/VCFS region.

De novo proximal interstitial deletions of 14q: cytogenetic and molecular investigations.

We report on 2 unrelated patients who had chromosome analysis performed because of psychomotor delay, failure to thrive, and minor anomalies. Each patient had a novel proximal 14q deletion (q11.2 to q21.1 in patient 737 and q12 to q22 in patient 777). Polymorphic (C-A)n microsatellite markers distributed along the length of chromosome 14q were examined in both patients and their parents in order to determine which marker loci were deleted. The deletion in patient 737 was found to be paternal in origin, based on the analysis of 2 marker loci (D14S54 and D14S70), thus assigning these loci to the deleted interval q11.2 q21.1. Furthermore, 3 loci were not deleted (TCRD, D14S50, and D14S80), suggesting that they are within or proximal to 14q11.2. In the other family (patient 777), none of the markers were fully informative, but the deleted chromosome was determined to be paternally derived based on cytogenetic heteromorphisms. Despite having overlapping proximal 14q deletions, these 2 patients shared few phenotypic similarities except for failure to thrive, micrognathia, and hypoplasia of the corpus callosum. Therefore, a distinct proximal 14q deletion syndrome is not yet apparent. However, the molecular analyses facilitated the localization of several 14q DNA markers to the deletion regions in these 2 patients, while excluding other markers from each deletion.

Normal expression of the Fanconi anemia proteins FAA and FAC and sensitivity to mitomycin C in two patients with Seckel syndrome.

Seckel syndrome is a rare autosomal recessive disorder. The classical presentation includes pre- and postnatal growth deficiency, mental retardation, and characteristic facial appearance. There have been several reports of associated hematological abnormalities and chromosomal breakage, findings suggestive of Fanconi anemia (FA). We tested for these findings in two Arabic patients with this syndrome. We compared the growth profile of lymphoblastoid cells from our patients and their parents with the FA group A cell line HSC72 in the presence and absence of mitomycin C (MMC). By Western analysis, we also determined the expression of FAA and FAC, two FA disease gene products that together account for approximately 80% of FA. Unlike HSC72 cells, cells from the patients were resistant to MMC, and both FAA and FAC proteins were expressed at similar levels in all cell lines. There is an increasing recognition of clinical variability and perhaps genetic heterogeneity in Seckel syndrome. Our results demonstrate that cross-link sensitivity comparable to FA is not a uniform finding in patients with Seckel syndrome.

Inherited duplication Xq27-qter at Xp22.3 in severely affected males: molecular cytogenetic evaluation and clinical description in three unrelated families.

We describe the clinical phenotype in four males from three families with duplication (X)(qter-->q27::p22.3-->qter). This is an unusual duplication of the distal long arm segment, Xq27-qter, onto the distal short arm of the X chromosome at Xp22.3, as shown by fluorescent in situ hybridization analysis with multiple X-specific probes. The patients are young male offspring of three unrelated, phenotypically normal carrier women. The affected males have similar clinical manifestations including severe growth retardation and developmental delay, severe axial hypotonia, and minor anomalies. Such clinical similarity in three unrelated families demonstrates that this chromosome abnormality results in a new and distinct clinical phenotype. Replication studies, performed on two of the mothers, provided evidence that inactivation of the abnormal X chromosome permitted the structural abnormality to persist in these families for a generation or more in females without phenotypic expression.

Mitochondrial DNA depletion associated with partial complex II and IV deficiencies and 3-methylglutaconic aciduria.

We report a patient with mitochondrial DNA depletion, partial complex II and IV deficiencies, and 3-methylglutaconic aciduria. Complex II deficiency has not been previously observed in mitochondrial DNA depletion syndromes. The observation of 3-methylglutaconic and 3-methylglutaric acidurias may be a useful indicator of a defect in respiratory chain function caused by mitochondrial DNA depletion.

An update on chromosome deletion and microdeletion syndromes.

Chromosome deletion and microdeletion syndromes account for an increasing number of clinically recognizable genetic conditions. New deletion syndromes continue to be characterized, and a number of previously described syndromes are being found to be due to chromosomal deletions or microdeletions. Fluorescent in situ hybridization technologies are in wide clinical use to diagnose deletion and microdeletion syndromes, and future uses of these technologies will provide prognostic information for patients and their parents, as the genes responsible for the phenotypic aspects of various deletion syndromes are identified. Future research studies will focus on delineating critical deletion intervals at a molecular level, and identifying candidate genes for the phenotypic features of deletion and microdeletion syndromes, toward the goal of understanding the pathology of the abnormal developmental and physiologic processes involved in each syndrome.

The use of genetic complementation in the study of eukaryotic macromolecular evolution: rate of spontaneous gene duplication at two loci of Drosophila melanogaster.

Gene duplications must play an important role in the evolutionary development of living organisms. Presented here is a general scheme that uses complementary alleles to isolate gene duplications in diploid organisms. The technique was used in Drosophila melanogaster to assess the rate of spontaneous gene duplication at two loci, maroon-like and rosy. The results indicate that the rate of duplication of the maroon-like locus is on the order of 2.7 X 10(-6); that the rate of duplication of the rosy locus is approximately 1.7 X 10(-4); and that duplication occurs in males, suggesting that there may actually be two modes of gene duplication in Drosophila melanogaster.

Unknown syndrome in sibs: pili torti, growth delay, developmental delay, and mild neurological abnormalities.

We present male and female sibs of consanguineous parents with features including pili torti with unusual hair shaft electron microscopic (EM) findings, growth delay, developmental delay, and mild to moderate neurological abnormalities. The features of the cases presented here have not been noted in the previously reported clinical syndromes in which pili torti may be found.

Monosomy 1p36.

We have reviewed published reports on patients with segmental aneusomy for chromosome 1p36 to help geneticists and other health professionals in the recognition of this emerging chromosomal syndrome. Terminal deletions of the short arm of chromosome 1 are associated with hypotonia and developmental delay (usually severe), growth abnormalities (growth retardation, microcephaly, obesity), and craniofacial dysmorphism with a large anterior fontanelle, prominent forehead, deep set eyes, flat nasal bridge and midface hypoplasia, ear asymmetry, a pointed chin, and orofacial clefting. Minor cardiac malformations, cardiomyopathy, seizures, and ventricular dilatation are the more common additional findings. Sensorineural hearing loss and variable ophthalmological anomalies have also been frequently observed. Although the deletions can be detected by high resolution cytogenetic studies, confirmation by fluorescence in situ hybridisation is required in most cases. The majority of deletions are maternally derived. Molecular characterisation of 1p36 deletions has been undertaken in several cases, and it is likely that this condition is a contiguous gene deletion syndrome.

Fine mapping of mutations in the fusion-inducing MP strain of herpes simplex virus type 1.

Previous studies [W. T. Ruyechan, L. S. Morse, D. M. Knipe, and B. Roizman (1979) J. Virol. 29, 677-697] have shown that multiple mutations are responsible for the mutant phenotypes of herpes simplex virus type 1, strain MP, and have indicated that these mutations may be located on the genome between map coordinates 0.70 and 0.83. Strain MP produces large syncytial (Syn) plaques on many cell types and does not express glycoprotein C (gC-), whereas a sibling strain mP produces wild-type, small, nonsyncytial (Syn+) plaques and is gC+. Cloned DNA fragments from strains MP and mP (and strain F) were used in marker transfer and marker rescue experiments to map more precisely the mutations in MP. It was found that a 680-bp fragment from MP DNA (map coordinates 0.735 to 0.740) could transfer a Syn marker to mP and that, conversely, an overlapping fragment from mP DNA (map coordinates 0.728 to 0.744) could rescue the Syn mutation of MP. Recombinant viruses obtained in these experiments differed from the donor of the cloned DNA fragment in plaque size, however, indicating that mutation(s) at other regions of the MP genome cause enlarged plaques, in which the infected cells are less rounded than in wild-type plaques. A fragment of MP DNA from map coordinates 0.60 to 0.64 transferred a mutation causing the gC- phenotype to strain mP, and a fragment of F DNA from map coordinates 0.62 to 0.64 rescued the gC- mutation of MP. These results, coupled with data published by Frink et al. [(1983) J. Virol. 45, 643-467], indicate that the mutation responsible for the gC- phenotype of MP may be in the structural gene for gC.

Molecular and clinical characterization of a patient with duplication of 1p36.3 and metopic synostosis.

Chromosome 1p duplications are rare. There have been only 11 reported cases of isolated 1p duplication, all of which were proximal, interstitial duplications. We present a patient with a terminal duplication of 1p (1p36.3). To our knowledge, this is the first such reported case. Our patient presented with metopic synostosis, rectal stenosis, atrial septal defect, and mildly delayed gross motor development. Molecular characterization using microsatellite marker analysis and fluorescence in situ hybridization (FISH) revealed an area of duplication between p58 and D1S2893, approximately 13 cM in size. We compare our patient's clinical findings with the clinical phenotype found in patients with the corresponding deletion of 1p36.3 and discuss the role of gene dosage in other deletion/duplication syndromes.