[Clinical parameters for molecular testing of Maturity Onset Diabetes of the Young (MODY)]. / Molekulargenetische Diagnostik bei Verdacht auf Maturity Onset Diabetes of the Young (MODY): Klinische Parameter zur Entscheidungshilfe.

BACKGROUND: Monogenic forms of diabetes are often diagnosed by chance, due to the variety of clinical presentation and limited experience of the diabetologists with this kind of diabetes. Aim of this study was to evaluate clinical parameters for an efficient screening. METHODS: Clinical parameters were: negative diabetes-specific antibodies at onset of diabetes, positive family history of diabetes, and low to moderate insulin requirements after one year of diabetes treatment. Molecular testing was performed through sequencing of the programming regions of HNF-4alpha (MODY 1), glucokinase (MODY 2) and HNF-1alpha/TCF1 (MODY 3) and in one patient the HNF-1beta/TCF2 region (MODY 5). 39 of 292 patients treated with insulin were negative for GADA and IA2A, and 8 (20.5%) patients fulfilled both other criteria. RESULTS: Positive molecular results were found in five (63%) patients (two with MODY 2, two with MODY 3, one with MODY 5). At diabetes onset, the mean age of the 5 patients with MODY was 10.6 ± 5.3 yrs (range 2.6-15 yrs), HbA(1c) was 8.4 ± 3.1 % (6.5-13.9%), mean diabetes duration until diagnosis of MODY was 3.3 ± 3.6 yrs (0.8-9.6 yrs) with insulin requirements of 0.44 ± 0.17 U/kg/d (0.2-0.6 U/kg/d). Patients with MODY 3 were changed from insulin to repaglinide, those with MODY 2 were recommended discontinuing insulin treatment. CONCLUSION: In patients with negative diabetes-specific antibodies at onset of diabetes, with a positive family history, and low to moderate insulin needs a genetic screening for MODY is indicated. Watchful consideration of these clinical parameters may lead to an early genetic testing, and to an adequate treatment.

New PCR assay for rapid and quantitative detection of human cytomegalovirus in cerebrospinal fluid.

Rapid Chelex extraction combined with an automated hybridization assay for the detection of PCR-amplified human cytomegalovirus DNA from cerebrospinal fluid was established. Quantitation of DNA was performed with a plasmid being used as an external standard. The detection limit was 10 copies per microliter. Quantitative detection of human cytomegalovirus DNA could be achieved over a range from 10 to 10(4) copies per microliter.

The HLA-DR4-associated DQw8 allele is confined to HLA-DR3/DR4 heterozygous type I (insulin-dependent) diabetics.

The HLA-DR4 specificity revealed a relative risk of 8.5 (chi 2 = 99.6; p less than 0.0001) when 193 type I diabetics were compared to 305 controls. Prevalence of the HLA-DR4-associated DQ types, i.e. DQw7 and DQw8, were determined, using a restriction fragment length polymorphism (RFLP) typing that combines the probe/enzyme combinations DQB/Taq I and DQB/Bam HI. The HLA-DQw8 specificity was confined to HLA-DR3/DR4 heterozygous patients when compared to controls (chi 2 = 4.9; p less than 0.025) or to all other DR4-heterozygous patients (chi 2 = 6.7; p less than 0.01). No association with HLA-DQw8 was seen in HLA-DR1/DR4 or HLA-DR"X"/DR4 (X not equal to 1,3,4) heterozygous patients. Due to the excess of HLA-DR3/DR4 patients the DQw8 allele is a risk factor in type I diabetics, but in HLA-DR1/DR4 and DRX/DR4 heterozygotes one might suggest that DQB1 and DRB combinations confer HLA-associated susceptibility.

Regional localization by in situ hybridization of a human chromosome 9 marker tightly linked to the Friedreich's ataxia locus.

In order to determine the regional localization of the Friedreich's ataxia (FA) gene on chromosome 9, the DNA probe DR47 (D9S5), which detects a restriction fragment length polymorphism (RFLP) in tight linkage with the disease, was hybridized in situ to metaphase chromosomes. Our results enable the D9S5 locus to be assigned to the 9q12-q13 region, thus indicating that this is also the localization of the FA gene.

Regional localisation of the Friedreich ataxia locus to human chromosome 9q13----q21.1.

We have previously assigned the Friedreich ataxia locus (FRDA) to chromosome 9; the current maximal lod score between FRDA and MCT112 (D9S15) is greater than 50 at a recombination fraction of theta = 0. The physical assignment of the locus defined by MCT112, and hence FRDA, has not been determined, although linkage analysis of MCT112 with other chromosome 9 markers inferred a location close to the centromere. We have used in situ hybridisation with MCT112, a corresponding cosmid MJ1, and DR47 (D9S5), coupled with mapping studies on hybrid cell panels, to define more precisely the location of the disease locus. The in situ location of all three probes is 9q13----q21.1, distal to the variable heterochromatin region. Physical assignment of FRDA will allow us to identify hybrid cell lines containing the mutated gene.

The Friedreich ataxia gene is assigned to chromosome 9q13-q21 by mapping of tightly linked markers and shows linkage disequilibrium with D9S15.

Chamberlain et al. have assigned the gene for Friedreich ataxia (FA), a recessive neurodegenerative disorder, to chromosome 9, and have proposed a regional localization in the proximal short arm (9p22-cen), on the basis of linkage to D9S15 and to interferon-beta (IFNB), the latter being localized in 9p22. We confirmed more recently the close linkage to D9S15 in another set of families but found much looser linkage to IFNB. We also reported another closely linked marker, D9S5. Additional families have now been studied, and our updated lod scores are z = 14.30 at theta = .00 for D9S15-FA linkage and z = 6.30 at theta = .00 for D9S5-FA linkage. Together with the recent data of Chamberlain et al., this shows that D9S15 is very likely within 1 cM of the FA locus. We have found very significant linkage disequilibrium (delta Std = .28, chi 2 = 9.71, P less than .01) between FA and the D9S15 MspI RFLP in French families, which further supports the very close proximity of these two loci. No recombination between D9S5 and D9S15 was found in the FA families or Centre d'Etude du Polymorphisme Humain families (z = 9.30 at theta = .00). Thus D9S5, D9S15, and FA define a cluster of tightly linked loci. We have mapped D9S5 by in situ hybridization to 9q13-q21, and, accordingly, we assign the D9S5, D9S15, and FA cluster to the proximal part of chromosome 9 long arm, close to the heterochromatic region.

Isolation and use of chromosome 1 probes for linkage studies on Charcot-Marie-Tooth disease.

Nine probes were isolated from a human chromosome 1 enriched library and mapped to regions of chromosome 1 using somatic cell hybrid lines. One clone, LR67, which mapped to 1q12----q23 detected a BglI RFLP. This probe, as well as 4 other known chromosome 1 markers, alpha-spectrin, Factor XIIIB, DR10 and DR78, were used for linkage studies in 15 Charcot-Marie-Tooth disease (CMT1) families. Close linkage of CMT1 to any of the 5 markers was not indicated. Total lod scores excluded linkage of CMT1 to LR67 and to DR10 at 5 cM or less, to DR78 at 10 cM or less, alpha-spectrin at 15 cM or less and Factor XIIIB at 20 cM or less. Possible linkage, however, was shown between LR67 and CMT1 at a distance of 30 cM. Also linkage at a distance of 5 cM was detected between this probe and alpha-spectrin.

Prenatal diagnosis of cystic fibrosis.

Cystic fibrosis is the most common autosomal recessive genetic disorder in the Caucasian population (1:2000-1:4000) (Warwick, W. J. (1978) Helv. Paediatr. Acta 33, 117-125). This defect is characterized by chronic obstructive pulmonary disease, pancreatic exocrine insufficiency and abnormally high perspiration electrolytes in most patients (Talamo et al. (1985) In: The metabolic basis of inherited diseases, pp. 1887-1917). The elevated electrolyte level provides the most reliable diagnostic test for cystic fibrosis homozygotes. Although prospects for cystic fibrosis patients have improved, genetically homozygous cystic fibrosis is effectively a lethal disease. Because of the seriousness of the disease, many families with one affected child desire a prenatal diagnosis when a second pregnancy occurs. Despite extensive research, the biochemical basis of cystic fibrosis remains unknown. Secondary effects on microvillar enzymes allow second trimester diagnosis (17-18 weeks of gestation (Brock, D. H. J. (1983) Lancet II, 941-943). First trimester prenatal diagnosis for cystic fibrosis became possible with DNA technology. Application of polymorphic marker loci to problems of prenatal diagnosis and carrier-testing is discussed.

Confirmation of linkage of Friedreich ataxia to chromosome 9 and identification of a new closely linked marker.

A linkage analysis with chromosome 9 markers was performed in 33 families with Friedreich ataxia (FA). Linkage with D9S15, previously established by S. Chamberlain et al. (1988, Nature London 334:248-249) was confirmed in our sample (z(theta) = 6.82 at theta = 0.02) while INFB (interferon-beta gene) shows looser linkage. An additional marker, D9S5, was also shown to be closely linked to FA (z(theta) = 5.77 at theta = 0.00).

Prenatal diagnosis of alpha-1-antitrypsin deficiency using oligonucleotide probe analysis.

Prenatal diagnosis of a pregnancy at risk for alpha-1-antitrypsin deficiency was performed by oligonucleotide probe analysis using M- and Z-specific oligonucleotides. The result was confirmed by the alternative approach utilizing restriction fragment length polymorphisms. Application of oligonucleotide analysis requires only fetal tissue if proteinase inhibitor types are accurately determined within the family. Our modified protocol is easy to carry out and is practicable in all laboratories where the Southern blot procedure has been established.

Evaluation of random cDNA clones as probes for human restriction fragment length polymorphisms.

We constructed two human cDNA libraries and selected clones hybridizing with more than five fragments of digested genomic DNA. We assume that these cDNAs detect sequences belonging to gene families. Compared with cDNAs derived from mRNAs of other tissues, the cDNAs of lymphocytes contained a higher proportion of these selected species of cDNA. We assume that these extra cDNAs are tissue-specific. In parallel tests, cDNAs belonging to gene families detected more restriction fragment length polymorphisms than did genomic probes, due to the larger number of restriction sites that can be checked using one probe. However, the chromosomal assignment of these polymorphisms often proved to be very difficult. In addition, we noticed that the mean length of EcoRI fragments hybridizing with our cDNAs is greater than the mean length of fragments hybridizing with randomly chosen genomic probes, possibly due to methylation connected with the inactivation of related active gene sequences.

Cystic fibrosis: typing 48 German families with linked DNA probes.

Two hundred and thirty five subjects from 48 German cystic fibrosis (CF) families were typed for restriction fragment length polymorphisms (RFLPs) detected by the probes pmet H, pmet D, and pJ 3.11, known to be tightly linked to the CF gene. Gene and haplotype frequencies suggest a linkage disequilibrium with the CF locus. The analysis of the predictive value of this typing in individual CF families indicates that the combined use of these probes provides a powerful diagnostic system both for carrier detection and prenatal diagnosis. In 33 out of 48 families carriers and non-carriers could be identified, and in 26 of these 33 families prenatal diagnosis could discriminate between affected and unaffected offspring.