Identification of four novel dinucleotide repeat polymorphisms in the IL-2 and IL-2beta receptor genes.

Two polymorphic regions have been described within the IL-2 and IL-2 receptor beta genes comprising 15 and 8 alleles, respectively. Whether these polymorphisms have biologic importance is unknown, although they have been variably identified in associated with certain chronic disease states. We report here the detection of four new alleles designated IL-2 A* (122 bp), IL-2R-2 (169 bp), IL-2R 0 (165 bp), and IL-2R 9 (147 bp) in patients with rheumatoid arthritis and normal controls from the Pacific Northwest. The number of alleles now recognized at these loci within the IL-2 and IL-2Rbeta genes increases to 16 and 12, respectively.

Association between dinucleotide repeat in non-coding region of interferon-gamma gene and susceptibility to, and severity of, rheumatoid arthritis.

BACKGROUND: Rheumatoid arthritis ranges from a mild, non-deforming arthropathy with little long-term disability to severe, incapacitating, deforming arthritis which may be refractory to conventional disease-modifying agents. Epidemiological studies show an important genetic influence in rheumatoid arthritis, and MHC region genes and cytokine genes within and outside this region have been considered as candidates. We did a case-control study to test whether polymorphisms in the interferon-gamma gene are associated with severity of rheumatoid arthritis. METHODS: Interferon gamma dinucleotide repeat polymorphisms were examined with quantitative genescan technology, and HLA-DR alleles were identified by PCR and restriction-fragment-length polymorphism analysis. We studied 60 patients with severe rheumatoid arthritis, 39 with mild disease, and 65 normal controls. FINDINGS: Susceptibility to, and severity of, rheumatoid arthritis were related to a microsatellite polymorphism within the first intron of the interferon-gamma gene. A 126 bp allele was seen in 44 (73%) of 60 patients with severe rheumatoid arthritis, compared with eight (21%) of 39 with mild disease (odds ratio 10.66 [95% CI 4.1-24.9]), and with eight (12%) of 65 normal controls (19.59 [7.7-49.9]). Conversely, a 122 bp allele at the same locus was found in four (7%) patients with severe disease compared with 25 (64%) of those with mild disease (0.04 [0.01-0.1]) and with 52 (80%) of controls (0.018 [0.005-0.06]). INTERPRETATION: This association may be valuable for understanding the mechanism of disease progression, for predicting the course of the disease, and for guiding therapy.

Identification of four novel dinucleotide repeat polymorphisms in the TNF-alpha and TNF-beta genes.

Five polymorphic regions (a to e) have been recognized within the TNF gene region. These polymorphisms appear to be of biological importance as individual alleles have been associated with higher production of TNF and/or an increased risk of rheumatoid arthritis or diabetes mellitus. We report here the detection of four new alleles designated a14 (122 bp), b8 (131 bp), b9 (132 bp), and d0 (122 bp) in patients with rheumatoid arthritis and normal controls from the Pacific Northwest. This increases to 39 the number of alleles now recognized at these loci.

Successful paternity of twins following bone marrow transplantation with busulfan, melphalan and cyclophosphamide conditioning.

A 33-year-old man who had received previous chemotherapy with cytarabine, daunorubicin and mitoxantrone followed by an autologous marrow transplant after conditioning with busulfan, melphalan and cyclophosphamide, fathered sex-mismatched fraternal twins approximately 6 years post-transplant. HLA and DNA analyses showed the probability of paternity to be in excess of 99% for each twin. To our knowledge this represents the first documented case of paternity following conditioning with this combination of marrow ablative agents and the first report of twin paternity following autologous marrow transplantation.

A novel DR beta 1*03 allele identified by polymerase chain reaction-restriction fragment length polymorphism.

This report describes a new MHC class II allele, HLA-DR beta 1*0306, discovered in a 31-year-old Norwegian male. The allele typed serologically as DRw52 (DR3) and amplified in PCR using DR52-associated group primers. This product could not be identified using established restriction digests, however. Use of Asp 700, Msp I, Hha I, Bse RI, Mnl I, Hph I, and Bsrb I gave banding patterns expected for a DR beta 1*03011 allele, but Rsa I had an additional site at codon 47. Sequencing showed a single base change at this position, with the substitution of tyrosine for phenylanine in this new allele. The biologic impact of this substitution remains to be determined.

Hemophilia B carrier determination based on family-specific mutation detection by DNA single-strand conformation analysis.

Single-strand conformation (SSC) analysis can distinguish normal from variant DNA fragments containing single point mutations by conformation-induced electrophoretic mobility shifts in non-denaturing polyacrylamide gels. We studied 25 hemophilia B kindreds by using SSC analysis after polymerase chain reaction (PCR) amplification of the eight factor IX exons and their intron boundaries. Variant SSC fragments were unambiguously identified in 24 kindreds, and direct DNA sequencing of variant PCR fragments identified 20 different hemophilia B mutations. This technique was used for rapid and accurate carrier determination in female family members without the need for additional sequencing studies, because carriers have both normal and hemophilia family-specific SSC fragments. Of 25 obligate carriers from 15 kindreds, 24 were confirmed to carry variant fragments. The exception, a patient's daughter homozygous for the normal allele, was demonstrated by subsequent PCR genotyping to be the result of non-paternity. In the additional 32 at-risk females from 16 kindreds studied, 19 were identified as carriers and 13 as non-carriers. Eleven of the unique mutations affected restriction enzyme digestion sites, and carriers could then be identified by appropriate restriction enzyme digestion of amplified DNA. Our study, with hemophilia B as a model system, demonstrates the accuracy and efficiency of SSC analysis in screening and tracking unknown mutations in monogenic inherited disorders with known gene sequences.

Brain dysgenesis and congenital intracerebral calcification associated with 3-hydroxyisobutyric aciduria.

Monozygotic male twins born to nonconsanguineous parents had dysmorphic facial features, microcephaly, migrational brain disorder, and congenital intracerebral calcification. They excreted excessive amounts of 3-hydroxyisobutyric acid, a metabolite of valine, and had evidence of impaired oxidative metabolism and metabolic acidosis. The level of 3-hydroxyisobutyrate in stored samples of midtrimester amniotic fluid was found to be high. The association of 3-hydroxyisobutyric aciduria with brain dysgenesis is a newly recognized mendelian disorder; its recurrence in a family at risk is potentially avoidable by prenatal diagnosis.

Hemophilia A carrier detection by restriction fragment length polymorphism analysis and discriminant analysis based on ELISA of factor VIII and vWf.

We performed carrier determination on female subjects from 32 hemophilia A kindreds with a combination of restriction fragment length polymorphism analysis and discriminant analysis of factor VIII antigen and von Willebrand factor antigen analyzed by enzyme-linked immunosorbent assay. Subjects included 25 obligate carriers, 30 at-risk female subjects from 19 kindreds each with two or more male subjects, with hemophilia and 28 at-risk female subjects from 13 kindreds each with a single sporadic case. Deoxyribonucleic acid (DNA) analysis with factor VIII intragenic probes clarified the carrier status of 15 female subjects, and extragenic probes classified an additional 14. Discriminant analysis, which identified 24 of 25 (96%) obligate carriers with carrier probabilities greater than or equal to 0.71 and 37 of 39 (95.0%) normal female subjects with probabilities less than or equal to 0.30, was useful for clarifying the carrier status of the female subjects who were not helped by DNA analysis and those that were classified by extragenic probes alone. Twenty-nine female subjects could not be categorized by restriction fragment length polymorphism analysis because DNA was unavailable from the subject or from key family members, key female subjects were noninformative, or carriership could not be excluded by restriction fragment length polymorphism in kindreds with sporadic cases. We studied 28 of these female subjects by discriminant analysis; 15 had carrier probabilities of greater than or equal to 0.71, 12 less than or equal to 0.30, and 1 = 0.35. All carriers by extragenic probes had carrier probability values greater than or equal to 0.71, whereas all noncarriers had values less than or equal to 0.30. In some families, particularly those in which gonadal mosaicism was a possibility, extensive family studies together with DNA and discriminant analyses were required for clarifying the source of the mutation, and hence the carrier status of the at-risk female subjects. Thus DNA and discriminant analyses complement each other, and when combined with careful pedigree analysis, the power of carrier determination is increased in some families.

Diagnosis of Duchenne and Becker muscular dystrophies by polymerase chain reaction. A multicenter study.

OBJECTIVE--To assess the efficiency, reliability, and ease of use of DNA diagnosis for Duchenne and Becker muscular dystrophies (DMD/BMD) using the polymerase chain reaction (PCR). DESIGN--DNA from the patients was screened for deletion mutations using multiplex PCR, and the results were compared with those obtained by Southern blot analysis. The PCR multiplex reaction detects nine specific "hot-spot" exons in the dystrophin gene while the Southern analysis detects 66 specific dystrophin gene restriction fragments. The multiplex reaction requires 50-fold less DNA than Southern analysis and thus is considerably more sensitive. SETTING--Fourteen university-affiliated and private genetic disease diagnostic laboratories. PATIENTS--Male patients with clinical signs of DMD/BMD. Cases were selected for analysis randomly, without knowledge of whether a deletion was present within the dystrophin gene. MAIN OUTCOME MEASURES--The percentage of cases that were detectable by multiplex PCR in comparison with Southern analysis, the frequency, extent, and location of the detected deletion mutations. In some cases, duplication mutations were monitored. RESULTS--The accuracy of a single PCR multiplex amplification (nine exons) was compared with Southern analysis with 10 cDNA probes that cover the full length of the gene. The multiplex PCR analytic method detected 82% of those deletions detected by Southern analysis methods. In one of 745 analyses, the multiplex method suggested a single exon deletion, which was not confirmed by Southern analysis, representing a false-positive rate of 0.013%. CONCLUSIONS--Multiplex PCR represents a sensitive and accurate method for deletion detection of 46% of all cases of DMD/BMD. The method requires 1 day for analysis, is easy to perform, and does not use radioactive tracers. As such, multiplex PCR represents an efficient and rapid method for prenatal or postnatal diagnosis of DMD/BMD.

Identification of factor IX mutations in haemophilia B: application of polymerase chain reaction and single strand conformation analysis.

The molecular characterization of two haemophilia B defects, Calgary 1 and Calgary 2, was carried out using polymerase chain reaction (PCR) amplification and direct dideoxy sequencing. It had been previously shown that the Calgary 1 mutation affects the 5' TaqI restriction site of exon VIII, whereas Calgary 2 involves the loss of the 3' TaqI site of exon VIII of the factor IX gene. Sequencing data has now revealed that each of these alterations involves a C-to-T transition within a CpG dinucleotide. In each instance an arginine residue is replaced by a stop codon. These cases represent the recurrence of each particular alteration, both of which are predicted to result in the production of a truncated protein lacking a significant part of the catalytic region. A recently developed technique that reveals base substitutions as single-strand conformation polymorphisms (SSCP) was adapted for modelling in the detection of point mutations. Referred to here as single-strand conformation (SSC) analysis, this procedure, used in association with PCR, provided a reliable and sensitive system for molecular diagnosis in each of the cases presented. Computer-generated secondary structure predictions demonstrated a strong correlation with experimental results and the technique was used to screen 11 additional patients in the same region. A change detected by SSC analysis in one patient was localized to 55 base pairs, sequenced, and identified as a conservative amino acid substitution. This patient is now referred to as Calgary 3.

Tentative assignment of gene for oto-palato-digital syndrome to distal Xq (Xq26-q28).

Detailed physical mapping of oto-palato-digital (OPD) syndrome gene on the X-chromosome was attempted on a family of 3 generations with 2 affected men. Although the result remains statistically non-significant, it indicates that the OPD-I gene might be located on the distal Xq.

Severe combined immune deficiency presenting with cyclic hematopoiesis.

At age 2 months a male infant presented with a cyclic clinical syndrome every 14-21 days that included pharyngeal aphthous ulcers, high fever, lymphadenopathy, pallor, and malaise. Serial blood studies indicated cycling of all blood cell elements, compatible with a diagnosis of cyclic hematopoiesis (CH). He also manifested a progressively severe immune deficiency, not described before in human CH. When first studied at age 5 months, he was hypogammaglobulinaemic with normal B lymphocyte numbers. By 6.5 months, he was agammaglobulinaemic. At age 8 months, he developed severe pneumocystis carinii pneumonia, and studies showed a state of severe combined immune deficiency. The patient received a bone marrow transplant from his HLA-identical sister with no preconditioning therapy. Subsequently, normal immune function developed and the cyclic hematopoiesis resolved. The majority of lymphocytes is of donor origin. Persistence of erythrocytes and neutrophils of recipient origin suggests that the hematopoietic stem cells were not abnormal. We speculate that this patient had a primary deficiency of a differentiation factor affecting maturation of lymphoid and myeloid progenitor cells.

Genetic stability of the D1Z2 region: implications for DNA genotyping and paternity testing.

The aim of the present study was to examine a single locus variable number tandem repeat for the purpose of DNA genotyping ("fingerprinting"). DNAs of 175 individuals from five ethnic groups (Black, Chinese, Japanese, Caucasian, and Melanesian) were analyzed. Restriction fragment length polymorphic analysis of random individuals revealed individual specific DNA patterns in all but one group. Among 20 Melanesian inhabitants of the Vanuatu islands in the southwest Pacific, three individuals were found to share a common pattern. This island population represents a "genetic isolate" and illustrates the importance of carrying out population studies on individual ethnic groups of interest. The complexity and the genetic stability of the D1Z2 region as revealed by the probe hMF1 make it an excellent candidate for DNA genotyping in paternity testing as 101 Caucasian individuals each had unique patterns for PstI and SinI digests.

Kinetochore analysis of micronuclei allows insights into the actions of colcemid and mitomycin C.

We have induced micronuclei in two strains of diploid human fibroblasts with a known aneugen, colcemid, and a known clastogen, mitomycin C. Using immunofluorescence to detect the presence of kinetochores in micronuclei, we were able to demonstrate a 26.8-fold increase in fluorescence-positive micronuclei (aneuploidy) in colcemid-treated cells. However, colcemid also induced an increase in kinetochore-negative micronuclei. Our findings support previous reports that suggest colcemid may induce chromosome breakage in addition to its major aneugenic effect. The frequency of kinetochore-negative micronuclei (chromosome breakage) in mitomycin C-treated cells rose an average of 7.9-fold in the two test strains, a clear reflection of its clastogenic action. However, a 4-fold increase in the kinetochore-positive fraction was seen. We conclude that the fibroblast micronucleus assay, coupled with kinetochore immunofluorescence, provides a useful screening approach for genotoxic agents. The delineation of the precise mechanism by which an agent perturbs the rates of chromosomal breakage or lag may require more detailed analysis.

Pallister-Killian syndrome: additional manifestations of cleft palate and sacral appendage.

We report a case of Pallister-Killian syndrome in a 28 week gestation infant. In addition to the characteristic phenotype, this patient had a cleft palate, diaphragmatic hernia, sacral appendage, and imperforate anus. The lymphocyte karyotype showed 96% 46,XX/4% 47,XX+i (12p) and the fibroblast karyotype 47,XX,+marker (presumed i(12p]. Fibroblast cytogenetic studies should be considered in all cases of diaphragmatic hernia associated with other malformations.

A familial interstitial deletion of the long arm of chromosome 21.

A mother and daughter with an interstitial deletion of the chromosome segment 21q11 to 21q21.3 have similar minor dysmorphism and mild mental retardation. These two patients are compared to others in the literature with deletion of the same region of chromosome 21. Molecular analysis of DNA from our patients localizes the DNA segments D21S1, D21S11, D21S8, and D21S22 within the deleted region.