Senescent BALB/c mice exhibit decreased expression of lambda5 surrogate light chains and reduced development within the pre-B cell compartment.

Although senescent BALB/c mice (approximately 2 years old) have reduced numbers of small pre-B cells, early pre-B cells (CD43+CD25+B220+) are present in comparable numbers within the bone marrow of both young (3-6-month-old) and senescent BALB/c mice. The transition of CD43+ pre-B cells to the CD43- pre-B cell compartments is dependent on proliferation and clonal maturation dictated by the pre-B cell receptor (mu/lambda5/VpreB). In vivo, senescent CD43+B220+ pro-B/early pre-B cells demonstrated reduction of lambda5 mRNA, by RT-PCR analysis, and of both surface and cytoplasmic lambda5 protein. Decreased lambda5 protein expression was also seen among pro-B/pre-B cells derived from senescent bone marrow after stimulation in vitro with IL-7. We propose that diminished expression of the lambda5 surrogate light chain results in decreased pre-B cell receptor formation and contributes to reduced recruitment of nascent CD43+ pre-B cells into the CD43- large and small pre-B cell compartments.

Congenital erythropoietic porphyria: identification and expression of 10 mutations in the uroporphyrinogen III synthase gene.

To investigate the molecular basis of the phenotypic heterogeneity in congenital erythropoietic porphyria, the mutations in the uroporphyrinogen III synthase gene from unrelated patients were determined. Six missense (L4F, Y19C, V82F, V99A, A104V, and G225S), a nonsense (Q249X), a frameshift (633insA), and two splicing mutations (IVS2+1 and IVS9 delta A + 4) were identified. When L4F, Y19C, V82F, V99A, A104V, 633insA, G225S, and Q249X were expressed in Escherichia coli, only the V82F, V99A, and A104V alleles expressed residual enzymatic activity. Of note, the V82F mutation, which occurs adjacent to the 5' donor site of intron 4, resulted in approximately 54% aberrantly spliced transcripts with exon 4 deleted. Thus, this novel exonic single-base substitution caused two lesions, a missense mutation and an aberrantly spliced transcript. Of the splicing mutations, the IVS2+1 allele produced a single transcript with exon 2 deleted, whereas the IVS9 delta A+4 allele was alternatively spliced, approximately 26% being normal transcripts and the remainder with exon 9 deleted. The amount of residual activity expressed by each allele provided a basis to correlate genotype with disease severity, thereby permitting genotype/phenotype predictions in this clinically heterogeneous disease.

Hydroxymethylbilane synthase: complete genomic sequence and amplifiable polymorphisms in the human gene.

Acute intermittent porphyria (AIP), an autosomal dominant inborn error of heme biosynthesis, results from the half-normal activity of the heme biosynthetic enzyme hydroxymethylbilane synthase (HMB-synthase). Heterozygous individuals are prone to life-threatening acute neurologic attacks, which are precipitated by certain drugs and other metabolic, hormonal, and nutritional factors. Since the biochemical diagnosis of heterozygous individuals has been problematic, recent efforts have focused on the identification of mutations and diagnostically useful restriction fragment length polymorphisms (RFLPs) in the HMB-synthase gene. To facilitate these endeavors, the human HMB-synthase gene, including 1.1 kg of the 5' flanking region, was isolated and completely sequenced in both orientations. The 10,024-bp gene contained 15 exons ranging in size from 39 to 438 bp and 14 introns ranging from 87 to 2913 bp. All intron/exon boundaries conformed to the GT/AG consensus rule. There were six Alu repetitive elements, one of the J and five of the Sa subfamilies. Analysis of the 1.1-kb 5' flanking region revealed putative regulatory elements for the housekeeping promoter including AP1, AP4, SP1, TRE, ENH, and CAC. This region contained 10 HpaII sites and had an overall GC content of 54%. Intron 1, which contained the erythroid-specific promoter, had putative regulatory motifs for NF-1, NF-E1, NF-E1(b), NF-E2, AP1, AP4, TOPO, CAAC, CAC, CAAT, and TATA. The locations and variant nucleotides for the known RFLPs in intron 1 were identified [MspI, nucleotide 1345 G/A; PstI, 1500 C/T; ApaLI, 2377 C/A; and BstNI, 2479 G/A] and improved polymerase chain reaction (PCR)-based detection methods for each were established.(ABSTRACT TRUNCATED AT 250 WORDS)

Congenital erythropoietic porphyria. A mild variant with low uroporphyrin I levels due to a missense mutation (A66V) encoding residual uroporphyrinogen III synthase activity.

BACKGROUND AND DESIGN: Congenital erythropoietic porphyria, an inborn error of heme biosynthesis, results from the deficient activity of the enzyme uroporphyrinogen III synthase. The clinical manifestations in unrelated patients with this autosomal recessive disorder are remarkedly variable, ranging from mild cutaneous involvement to severe transfusion-dependent hemolytic anemia. Biochemical and molecular studies were undertaken to investigate the nature of the unusually mild phenotype in a 15-year-old boy with only cutaneous manifestations. RESULTS: The proband's levels of total porphyrins, urinary uroporphyrin I, and erythrocyte coproporphyrin I were elevated, but not as dramatically as in other patients with this porphyria. Interestingly, the erythrocyte uroporphyrinogen III synthase activity in the proband was about 21% of the normal mean, indicating the presence of significant residual activity. In cultured lymphoblasts from the proband, his father, and mother, the enzymatic activities were 10%, 70%, and 50% of the normal mean, respectively. Molecular analyses revealed that the proband was heteroallelic for two uroporphyrinogen III synthase missense mutations: the C73R allele inherited from his mother and the A66V allele transmitted by his father. The A66V allele encoded residual enzymatic activity in vitro while the C73R allele did not. CONCLUSIONS: The A66V allele accounted for the proband's low levels of porphyrin accumulation and mild clinical manifestations. Such genotype-phenotype correlations should provide understanding of the remarkable clinical variability in other patients with this inherited porphyria.

Subcutaneous pancreatic fat necrosis associated with acute arthritis.

Subcutaneous fat necrosis is a well described, rare sequela of acute pancreatitis. Uncommonly, arthritis is seen in association with these 2 disease processes. We report a case of fulminant pancreatitis presenting as an acute arthritis. Birefringent crystal-like structures led to initial diagnostic confusion with gout.

Congenital erythropoietic porphyria: identification and expression of exonic mutations in the uroporphyrinogen III synthase gene.

Congenital erythropoietic porphyria (CEP), an inborn error of heme biosynthesis, results from the deficient activity of uroporphyrinogen III synthase (URO-synthase). This autosomal recessive disorder is heterogeneous; patients with severe disease are often transfusion dependent, while milder patients primarily have cutaneous involvement. To investigate this phenotypic heterogeneity, exonic point mutations in the URO-synthase gene were identified in unrelated CEP patients. Four missense mutations were identified: (a) an A to G transition of nucleotide (nt) 184 that predicted a Thr to Ala substitution at residue 62 (designated T62A); (b) a C to T transition of nt 197 that encoded an Ala to Val replacement at residue 66 (A66V); (c) a T to C transition of nt 217 that predicted a Cys to Arg substitution at residue 73 (C73R); and (d) a C to T transition of nt 683 that resulted in a Thr to Met replacement at residue 228 (T228M). In addition, a G to A transition of nt 27 that did not change the encoded amino acid (A9A) was detected in an African patient. The T62A, C73R, and T228M alleles did not express detectable enzymatic activity, while the A66V allele expressed residual, but unstable activity. The C73R allele was present in eight of 21 unrelated CEP patients (21% of CEP alleles). In three patients, identification of both alleles permitted genotype-phenotype correlations; the A66V/C73R, T228M/C73R, and C73R/C73R genotypes had mild, moderately severe, and severe disease, respectively. These findings provide the first genotype-phenotype correlations and permit molecular heterozygote detection in this inherited porphyria.

Regional assignment of the human uroporphyrinogen III synthase (UROS) gene to chromosome 10q25.2----q26.3.

Uroporphyrinogen III synthase [UROS; hydroxymethylbilane hydro-lyase (cyclizing), EC 4.2.1.75] is the fourth enzyme in the human heme biosynthetic pathway. The recent isolation of the cDNA encoding human UROS facilitated its chromosomal localization. Human UROS sequences were specifically amplified by the polymerase chain reaction (PCR) from genomic DNA of two independent panels of human-rodent somatic cell hybrids. There was 100% concordance for the presence of the human UROS PCR product and human chromosome 10. For each of the other chromosomes, there was 19%-53% discordance with human UROS. The chromosomal assignment was confirmed by Southern hybridization analysis of DNA from somatic cell hybrids with the full-length UROS cDNA. Using human-rodent hybrids containing different portions of human chromosome 10, we assigned the UROS gene to the region 10q25.2----q26.3.

DNA polymorphism of major histocompatibility complex class II and class III genes in systemic lupus erythematosus.

We investigated the Taq I digested DNA restriction fragment length polymorphism (RFLP) of the Major Histocompatibility Complex (MHC) class II genes: HLA-DRB, -DQA, and the class III genes: C4 and 21-hydroxylase(CYP21) in 56 caucasoid patients with systemic lupus erythematosus (SLE) and 62 control subjects in order to define the molecular variation of these genes and their association with SLE. The results showed that the gene frequencies of both HLA-DR2 and -DR3 were significantly increased in the SLE population compared to normal subjects (DR2: 21.4% vs 10.7% chi 2 = 4.5. DR3: 29.6% vs 13.3%; chi 2 = 8.3). A high frequency of C4A and CYP21A gene deletions was also found in SLE patients (SLE 52%, normals 24%). All of 22 SLE patients, and 12 of 15 normal subjects who had C4A and CYP21A gene deletions had a 10.0kb Taq 1 DRB RFLP attributable to the presence of HLA-DR3. Family studies showed linkage of C4A/CYP21A deletions with HLA-B8 and -DR3, and confirmed the previously demonstrated association of the HLA-B8, DR3, C4A*Q0, C4*B1, Bf*S, C2*C haplotype with SLE. Deletions affecting the C4A and CYP21A genes were the commonest cause of C4A null alleles in SLE. No strong association between C4 null phenotype or C4 gene deletion, as determined by RFLP, was observed in patients who possessed DR2.

Screening for cervical carcinoma.

At present, cytological examination of cervical smears is the method used to screen the female population for cervical abnormalities. This employs the Papanicolaou staining technique and cell morphology to detect abnormal cells from the susceptible transformation zone of the cervix. However, with the recent discovery of a possible link between human papillomavirus (HPV) infection and cervical cancer, a test to determine the presence of HPV infection may be appropriate. Tests are still at a developmental stage and at present used only for research purposes. Various DNA-DNA or DNA-RNA hybridisation techniques are employed in experimental screening studies, and these involve probing cells or DNA extracted from cells for HPV DNA using radio-labelled probes.

Nursing mothers' wrist - stenosing tenovaginitis of De Quervain - abstract

Fifty-two patients treated for painful wrists during a seven-year period at a referral clinic were studied. Forty-seven were women, and of these, twenty-seven were nursing mothers. This presentation shows the results of an investigation into some of the factors in the aetiology of this condition. Handling of the baby, especially those of higher birth weight, may be a contributing factor in the production of the disease. Conservative treatment by injection of local anaesthetic and hydrocortisone mixture into the tendon sheath is successful in early cases. Late cases benefit from operative relief of the stenosis (AU)

DQ beta polymorphism and genetic susceptibility to Felty's syndrome.

We used an oligonucleotide probe specific for a polymorphic sequence in the HLA-DQ beta gene to investigate the role of DQ polymorphism in genetic susceptibility to Felty's syndrome (FS) and rheumatoid arthritis (RA). The sequence of this gene was identified from a complementary DNA library derived from an RA patient's B lymphoblastoid cell line. With this probe, we studied the prevalence of the specific DQ beta allele in DR4 positive FS patients, RA patients, and normal control subjects. Significantly more FS patients (17 of 25) showed hybridization with this oligonucleotide probe, compared with the number of DR4 positive non-FS RA patients (7 of 23) and normal controls (7 of 21). The findings indicate that genes linked to the DQ region are important in determining susceptibility to FS.

Immunogenetics of Graves' ophthalmopathy.

We have performed an immunogenetic analysis of 53 patients with severe Graves' ophthalmopathy, 51 patients with Graves' disease but little or no clinically apparent eye disease, and 90 controls. The distribution of restriction fragment length polymorphisms was analysed in the three groups, using probes for the HLA-DQ alpha and DR beta regions, the T-cell receptor C alpha, V alpha, C beta and J gamma genes and the immunoglobulin gene switch regions, S alpha and S mu. There was no abnormal distribution of these polymorphisms in either group of Graves' patients, or differences between the Graves' patients with or without eye disease. It was possible to assign HLA-DR types in most patients using the polymorphisms found after probing with DQ alpha and DR beta; there was no abnormal distribution of DR types (including HLA-DR3) assigned by restriction fragment polymorphisms in the two Graves' groups. These results fail to confirm the reported associations between ophthalmopathy and HLA-DR3 and between Graves' disease and the T-cell receptor C beta polymorphism; they also argue against a strong influence of Gm allotypes in Graves' disease since these genes are in linkage disequilibrium with the S alpha polymorphisms. The association of Graves' disease with HLA-DR3, defined hitherto using serological reagents, may be less strong than previously described.