Atypical erythroblastosis in a patient with Diamond-Blackfan anemia who developed del(20q) myelodysplasia.

Diamond-Blackfan anemia (DBA) is a congenital red cell aplasia arising from ribosomal protein (RP) defects. Affected patients present with neonatal anemia, occasional dysmorphism, and cancer predisposition. An anemic newborn was diagnosed with DBA due to RPL5 mutation (c.473_474del, p.K158SfsX26). Refractory anemia required regular transfusions and iron chelation therapy. Pancytopenia occurred at age 16 years. Bone-marrow studies showed myelodysplasia, erythroblastosis, and clonal evolution of del(20)(q11.2q13.3). Severe anemia required transfusions. Del(20q), including the L3MBTL1 gene, is reported to be relevant to the hematological phenotype of Shwachman-Diamond syndrome. A combined defect of RPL5 and L3MBTL1 may contribute to the aberrant erythropoiesis in the present case.

Comparison of the pathologic and pathogenic features in six different regions of postmortem brains of three patients with fatal familial insomnia.

Fatal familial insomnia (FFI) is an autosomal dominant prion disease clinically characterized by rapidly progressive insomnia, prominent autonomic alterations and behavioral disturbance. The D178N mutation of the prion protein gene (PRNP) on chromosome 20 in conjunction with methionine at codon 129 is a molecular feature. Although the neuropathological characteristics of FFI are well documented, the neuropathologic and pathogenic features of FFI patients remain poorly understood. Six brain regions of postmortem brains from 3 FFI patients were examined using immunohistochemistry, western blot analyses and quantitative real-time PCR. In all 3 brain specimens, reactive astrogliosis was found to be more severe in the thalamus than in the cortex regions. Western blot analyses showed that all three brains expressed PrP, but only 2 were associated with significantly weak proteinase K (PK) resistance. However, the conformational stabilities of PrPSc in the 3 FFI brains were significantly weaker than those presented in a G114V genetic Creutzfeldt-Jakob disease (gCJD) case. Immunohistochemistry and western blot analyses showed comparable amounts of neuron-specific enolase (NSE)-positive stained cells and NSE protein among the different regions in the three brains. In addition, the transcriptional levels of glial fibrillary acidic protein (GFAP) and NSE-specific mRNAs were coincident with the expression of these proteins. In conclusion, in the present study, we described the detailed regional neuropathology of FFI cases.

Frontal motor seizure following non-convulsive status epilepticus in ring chromosome 20 syndrome.

The ring chromosome 20 syndrome is a rare syndrome characterized by intractable epilepsy with particular electro clinical features including episodes of prolonged confusional state and nocturnal frontal lobe seizures. We report a 17-year-old girl who had intractable epilepsy with frontal seizure and prolonged confusional state secondary to non-convulsive status epilepticus. The diagnosis of ring chromosome 20 was suspected and confirmed by karyotype. The cytogenetic study of CHRNA4 and KCNQ2 genes did not detect deletion in the ring chromosome 20. During video-EEG recording, this girl presented a non-convulsive status epilepticus that lasted more than 20 minutes followed by typical frontal lobe seizure. This association was not previously described, and was probably caused by chromosomal instability.

A novel splicing mutation in KCNQ2 in a multigenerational family with BFNC followed for 25 years.

PURPOSE: A large multigenerational family with benign familial neonatal convulsions (BFNC) was revisited to identify the disease-causing mutation and to assess long-term outcome. METHODS: We supplemented the original data with recent clinical and neurophysiologic data on patients and first-degree relatives, including information on seizure recurrence. We conducted linkage analysis at the EBN1 and EBN2 loci, followed by mutation analysis of KCNQ2. We evaluated the qualitative effect of the KCNQ2 mutation at the messenger RNA (mRNA) level by using reverse-transcribed total RNA isolated from leukocytes. RESULTS: Thirteen relatives had a history of neonatal convulsions, 11 of whom showed remission within 2 months. One patient showed an atypical course of neonatal convulsions, developing photosensitive myoclonic epilepsy at age 13 years. We found suggestive linkage of the BFNC phenotype to the 20q13-EBN1 locus (lod score, 2.03) and an intronic mutation IVS14-6 C>A in KCNQ2 segregating with the trait in all affected members, but absent in 100 unrelated control subjects. This mutation creates a new, preferentially used, splice site. Alternative splicing adds 4 nt containing a premature stop codon to the transcript, resulting in a truncated protein after position R588. CONCLUSIONS: We detected and characterized a novel splicing mutation in the brain-specific KCNQ2 gene by using easily accessible blood leukocytes. Aberrant splicing cosegregates with BFNC but not with photosensitivity.

Duplication of distal 20q: clinical, cytogenetic and array CGH. Characterization of a new case.

Trisomy of the long arm of chromosome 20 is rare. We describe an 18-month-old male who was born at 36 weeks via Caesarian section after an uneventful pregnancy. During the newborn period he was found to have a right-sided cleft lip and cleft palate, hypertelorism, strabismus and mildly over-folded ears with cupping. Cardiovascular examination was consistent with the diagnosis of severe aortic coarctation, which was confirmed by echocardiogram. Additionally, hypothyroidism was diagnosed. Neurological evaluation at 18 months revealed a hypotonic infant with delayed acquisition of motor milestones. Cytogenetic analysis showed additional material on the long arm of chromosome 20, confirmed by fluorescence in situ hybridization (FISH) analysis as being of chromosome 20 origin. Because of the indistinct GTG-banding pattern it was not possible to distinguish between a proximal [dup(20)(q11.2q13.1)] or distal duplication [dup(20)(q13.1q13.3)]. To further define the duplication we used array comparative genomic hybridization (CGH) which demonstrated a 7.8 Mb interstitial duplication in distal 20q. Thus, the proband's karyotype was interpreted as 46,XY,dup(20)(q13.2q13.2). The proband is the first reported case of a pure duplication of this region. This case further highlights the utility of array CGH in characterizing aneusomies and, in particular, for accurate breakpoint designation and quantitation of ambiguous rearrangements.

Identification of a novel human zinc finger protein gene ZNF313.

A novel human zinc finger protein gene that contains both ring finger and C(2)H(2) domain was first isolated by mRNA differential display between the testes of fertile adults and azoospermic patients followed by rapid amplification of cDNA ends (RACE). Total 6 exons of the human gene span a 17,484 bp genomic DNA sequence that was mapped to chromosome 20q13 by fluorescence in situ hybridization. The mature processed mRNA encodes a 228-amino acid protein with a C(3)HC(4) ring finger and three C(2)H(2) domains. Genomic analysis of the human gene identified two polyadenylation signals in exon 6 resulting in alternative 3'-untranslated regions. Results of Northern blot and RT-PCR of RNAs extracted from multiple tissues revealed that the gene has two transcripts of which the shorter transcript was expressed abundantly in fertile adult testes, but much less in testes of azoospermic patient, fetus as well as other human tissues. These data suggest that the gene may play a role in human spermatogenesis and male fertility.

Identification and characterization of novel mouse and human ADAM33s with potential metalloprotease activity.

The ADAM family of membrane-anchored proteins has a unique domain structure, with each containing a disintegrin and metalloprotease (ADAM) domain. We have isolated mouse and human cDNAs encoding a novel member of the ADAM family. The mouse and human predicted proteins consisted of 797 and 813 amino acids, respectively, and they shared 70% homology of the entire amino acid sequence. The mouse ADAM gene exists at a single gene locus. The human gene was ubiquitously expressed in tissues other than liver, was mapped to human chromosome 20p13, and was found to consist of 22 exons. Both proteins have domain organization identical to that of previously reported members of the ADAM family, and contain the typical zinc-binding consensus sequence (HEXGHXXGXXHD) in their metalloprotease domain and a pattern of cysteine localization (C(x)(3)C(x)(5)C(x)(5)CxC(x)(8)C) in their EGF-like domain that is typical of an EGF-like motif. The human protein shows homology with Xenopus ADAM13 (44%), human ADAM19 (40%), and human ADAM12 (39%). From the results of phylogenic analysis based on primary amino acid sequence and distribution of the mRNA, these novel ADAM genes were thus named ADAM33.

A novel conserved cochlear gene, OTOR: identification, expression analysis, and chromosomal mapping.

We have identified a novel cochlear gene, designated OTOR, from a comparative sequence analysis of over 4000 clones from a human fetal cochlear cDNA library. Northern blot analysis of human and chicken organs shows strong OTOR expression only in the cochlea; very low levels are detected in the chicken eye and spinal cord. Otor and Col2A1 are coexpressed in the cartilaginous plates of the neural and abneural limbs of the chicken cochlea, structures analogous to the mammalian spiral limbus, osseous spiral lamina, and spiral ligament, and not in any other tissues in head and body sections. The human OTOR gene localizes to chromosome 20 in bands p11.23-p12.1 and more precisely to STS marker WI-16380. We have isolated cDNAs orthologous to human OTOR in the mouse, chicken, and bullfrog. The encoded protein, designated otoraplin, has a predicted secretion signal peptide sequence and shows a high degree of cross-species conservation. Otoraplin is homologous to the protein encoded by CDRAP/MIA (cartilage-derived retinoic acid sensitive protein/melanoma inhibitory activity), which is expressed predominantly by chondrocytes, functions in cartilage development and maintenance, and has growth-inhibitory activity in melanoma cell lines.

Genomic organization, expression, and chromosome location of the human SNAIL gene (SNAI1) and a related processed pseudogene (SNAI1P).

Some of the zinc finger proteins of the snail family are essential in the formation of mesoderm during gastrulation and the development of neural crest and its derivatives. We have isolated the human SNAIL gene (HGMW-approved symbol SNAI1) and describe its genomic organization, having sequenced a region spanning more than 5882 bp. The human SNAIL gene contains three exons. The SNAIL transcript is 2. 0 kb and is found in placenta and adult heart, lung, brain, liver, and skeletal muscle. It codes for a protein of 264 amino acids and 29.1 kDa. This protein contains three classic zinc fingers and one atypical zinc finger. The human SNAIL protein is 87.5, 58.7, 50.9, 50.7, 55.4, and 31.5% identical to mouse Snail, chicken snail-like, zebrafish snail1, zebrafish snail2, Xenopus snail, and Drosophila snail proteins, respectively. The zinc finger region is 95.5% identical between human and mouse Snail. Because Drosophila snail and twist are important regulators during mesoderm development and because human TWIST mutations have been implicated in craniosynostosis, a cohort of 59 patients with craniosynostosis syndromes were screened for SNAIL mutations. None were found. By somatic cell and radiation hybrid mapping panels, SNAIL was localized to human chromosome 20q13.2, between markers D20S886 and D20S109. A SNAIL-related, putative processed pseudogene (HGMW-approved symbol SNAI1P) was also isolated and maps to human chromosome 2q33-q37.

A human nucleobase transporter-like cDNA (SLC23A1): member of a transporter family conserved from bacteria to mammals.

A family of related polytopic membrane proteins that mediate the transport of nucleobases has been extended to Homo sapiens by the cloning of a full-length human cDNA that encodes a nucleobase transporter-like protein. The protein is predicted to contain 11-14 transmembrane-spanning regions, exhibits 20-28% overall sequence identity to fungal and bacterial transporters, and contains a conserved signature motif found in this family. Fluorescence in situ hybridization localized the gene (HGMW-approved symbol SLC23A1) to human chromosome 20p13. Human nucleobase transporter-like mRNA was present in all tissues examined, with lower levels found in heart, skeletal muscle, and ovary. Expression of the 60-kDa cDNA-encoded protein was demonstrated by an in vitro transcription-translation approach. The identification of this nucleobase transporter-like protein will allow the further elucidation of the interaction of human cells with physiological nucleobases and pharmacologically important drugs such as 5-F-uracil, dideoxynucleosides, and acyclic nucleosides.

Identification and characterization of human MT5-MMP, a new membrane-bound activator of progelatinase a overexpressed in brain tumors.

A cDNA encoding a new member of the membrane-type (MT) matrix metalloproteinase (MMP) family has been identified and cloned from a human brain cDNA library. The isolated cDNA encodes a polypeptide of 645 amino acids that displays a similar domain organization as other MMPs, including a predomain with the activation locus, a zinc-binding site, and a hemopexin domain. The deduced amino acid sequence contains a COOH-terminal extension, rich in hydrophobic residues and similar in size to the equivalent domains identified in MT-MMPs. Immunofluorescence and Western blot analysis of COS-7 cells transfected with the isolated cDNA revealed that the encoded protein is localized in the plasma membrane. On the basis of these features, this novel human MMP has been called MT5-MMP because it represents the fifth member of the MT-MMP subfamily of MMPs. Fluorescent in situ hybridization experiments showed that the human MT5-MMP gene (MMP-24) maps to 20q11.2, a region frequently amplified in tumors from diverse sources. Northern blot analysis demonstrated that MT5-MMP is predominantly expressed in brain, kidney, pancreas, and lung. In addition, MT5-MMP transcripts were detected at high levels compared to normal brain tissue in a series of brain tumors, including astrocytomas and glioblastomas. The catalytic domain of MT5-MMP, produced in Escherichia coli as a fusion protein with glutathione S-transferase, exhibits a potent proteolytic activity against progelatinase A, leading to the generation of the Mr 62,000 active form of this enzyme. These data suggest that MT5-MMP may contribute to the activation of progelatinase A in tumor tissues, in which it is overexpressed, thereby facilitating tumor progression.

Frequency of trisomy 20 in nonmalignant bronchial epithelium from lung cancer patients and cancer-free former uranium miners and smokers.

Lung cancer is the leading cause of cancer-related deaths. The development of sensitive screening methods to identify at-risk individuals before emergence of clinical disease would permit early intervention that could decrease this mortality. Our previous studies have shown that cells with trisomy 7 can be detected in bronchial epithelium from cancer-free smokers and former uranium miners. However, the use of more than one molecular marker could increase the chance of identifying at-risk individuals. Trisomy 20, which is found in 43-57% of non-small cell lung cancers, is a candidate marker. The purpose of the current investigation was to determine the percentage of cells with trisomy 20 in persons with a high risk for lung cancer. Bronchial epithelial cells that had been assayed for trisomy 7 were assayed for trisomy 20 by fluorescence in situ hybridization. Trisomy 20 was detected in bronchial epithelial cells from lung cancer patients and from smokers and ex-uranium miners without lung cancer. In some cases, patients who were negative for trisomy 7 exhibited trisomy 20. Consequently, more people with field cancerization were identified using both markers. However, the two markers combined did not appear to stratify the risk for lung cancer.

Identification and cloning of the human homolog (JAG1) of the rat Jagged1 gene from the Alagille syndrome critical region at 20p12.

Notch proteins are a family of closely related transmembrane receptors proven to be instrumental in cell fate decisions. Recently, Notch ligands Delta and Jagged have been identified in Drosophila and rat, respectively. We have isolated the human homolog of the rat Jagged1 gene, JAG1, from a CpG island in a YAC clone covering the Alagille syndrome critical region at chromosome 20p12 (tel-SNAP-D20S186-cen). Alagille syndrome is an autosomal dominant disorder characterized by neonatal jaundice, paucity of intrahepatic bile ducts, and abnormalities of the heart, skeleton, and eyes. The human Jagged1 (JAG1), therefore, appears to be a strong candidate gene for this disease. Here we describe the identification, full-length cDNA cloning, expression patterns, and precise physical location of this gene within the Alagille syndrome critical region.