Linkage of microsatellites to the AGXT gene on chromosome 2q37.3 and their role in prenatal diagnosis of primary hyperoxaluria type 1

Defects in the AGXT gene mapped to chromosome 2q37.3 cause primary hyperoxaluria type 1 (PH1), one of the inherited disorders of endogenous oxalate overproduction. In order to identify diagnostically useful linkage markers in this region of chromosome 2 we have typed three microsatellite loci mapping to the q37 region of chromosome 2 in 192 individuals from 30 families. They were additionally studied for mutations and polymorphisms in the AGXT gene. Maximum lod scores of 29·1, 22·8 and 15·8 were obtained for D2S140, D2S125 and D2S395 respectively at recombination fractions (theta) of 0·001, 0·015 and 0·02. Confidence intervals for recombination as determined by the 'lod-1 rule' were 0·015, 0·05 and 0·06. Three recombinants were identified between AGXT and D2S125/D2S395, whereas no recombination between AGXT and D2S140 was observed. These data allow the calculation of the risk of incorrect prenatal diagnosis of PH1 based solely on linkage analysis with these extragenic markers.     

Very close linkage between D2S1 and ACP1 on chromosome 2p

The genomic DNA-probe L2.30 was used to assign D2S1 to 2p23–pter by in situ hybridization. The RFLP revealed by Bgl II was then used for linkage studies in the Oslo-NHIK families segregating for the acid phosphatase ACPl protein polymorphism. Evidence for very close linkage was found by a lod score of +17.17 at recombination fraction θ = 001. By this close linkage 92 informative meioses could be inferred from the families and with only a single crossover. The upper probability limit to the recombination fraction is 006 according to the HGM 8 criterion. No association between ACP1 alleles and D2S1 Bgl II alleles was found. The Norwegian gene frequencies for 0251 were A1 (9–0 kb) = 065 and A2 (6.3 kb) = 0.35.     

Charcot-Marie-Tooth neuropathy type 1A mutation: apparent crossovers with D17S122 are due to a duplication.

A locus for the slow conducting form of Charcot-Marie-Tooth neuropathy (CMT1A) was localised to the proximal short arm of chromosome 17, in band p11.2, distal to D17S58. Linkage studies of CMT1A in 3 large Australian families with the marker loci D17S58, D17S71, and D17S57 suggested the order, pter-CMT1A-D17S71-D17S58-centromere-D17S57. However, the estimate of the recombination fraction between CMT1A and D17S122, also assigned to p11.2, was incompatible with known map distances. The impasse was resolved when the D17S122 genotypes were revised to take into account a dosage effect due to a duplication. After correction of the genotypes, the maximum lod score between CMT1A and D17S122 increased from 0.53 at a recombination fraction of 0.3 to 34.28 at zero recombination. This result emphasizes that genotypes for markers in the p12-p11.2 region should be examined very carefully as ignoring the duplication changes the linkage results dramatically. The fact that no crossovers were found between CMT1A and D17S122 suggests that the duplication may cause the disease phenotype.     

Charcot-Marie-Tooth neuropathy type 1A mutation: Apparent crossovers with D17S122 are due to a duplication

A locus for the slow conducting form of Charcot-Marie-Tooth neuropathy (CMT1A) was localised to the proximal short arm of chromosome 17, in band p11.2, distal to D17S58. Linkage studies of CMT1A in 3 large Australian families with the marker loci D17S58, D17S71, and D17S57 suggested the order, pter–CMT1A–D17S71–D17S58–centromere–D17S57. However, the estimate of the recombination fraction between CMT1A and D17S122, also assigned to p11.2, was incompatible with known map distances. The impasse was resolved when the D17S122 genotypes were revised to take into account a dosage effect due to a duplication. After correction of the genotypes, the maximum lod score between CMT1A and D17S122 increased from 0.53 at a recombination fraction of 0.3 to 34.28 at zero recombination. This result emphasizes that genotypes for markers in the p12–p11.2 region should be examined very carefully as ignoring the duplication changes the linkage results dramatically. The fact that no crossovers were found between CMT1A and D17S122 suggests that the duplication may cause the disease phenotype. © Wiley-Liss, Inc.     

Linked polymorphic DNA markers in the prediction of X-linked muscular dystrophy

Ten polymorphic DNA markers, including gene specific markers of loci DXS164 and DXS206 , were tested for allele frequencies, degree of heterozygosity and linkage in 34 Finnish families with X-linked muscular dystrophy. With the exception of the Bam HI RFLP of DXS164 subclone pERT87-15, allele frequencies and the degree of heterozygosity failed to show any significant deviation from the data published elsewhere. We document a high degree of linkage disequilibrium between several RFLPs belonging to locus DXS164 . Our linkage data include one recombination between DMD and DXS164 enabling a tentative location of the mutation site distal to DXS164 . The maximum lod score for linkage between the disease locus and DX164 was 7.828 at a recombination fraction of 0.02. According to our data DXS28 and DXS43 may be located further away from the disease locus than previously thought. We use only gene specific markers for genetic counselling. Excluding deletions, 97.1 % of women were heterozygous for at least one such marker. A diagnostic procedure in which useful information can be obtained in over 90 % of all diagnostic situations, using only four filters, is proposed.     

Linkage studies in Menkes' disease The Xg blood group system and C-banding of the X chromosome

Menkes' disease is a rare, genetically determined disturbance of copper metabolism which is transmitted as an X-linked recessive character. By comparative gene mapping it can be suggested that the most likely localization of the gene for Menkes' disease is on the long arm of the human X chromosome close to band q l3. This regional assignment is supported by the present analysis of the genetic relationship between the Menkes locus, the Xg locus, and the centromere in five Danish families. The evidence suggests close linkage between the Menkes locus and the centromere. The most likely value of the recombination fraction is 0·05 and the maximum lod score is above the conventional +3 limit. Linkage analysis of the Menkes locus and the Xg locus showed a recombination value of 0·24, but the maximum conditional lod score is 0·28, which is far below the conventional +3 limit. The present study demonstrates a successful application of a chromosomal morphological marker in linkage analysis and carrier detection of a single gene disorder. The close linkage between the gene for Menkes' disease and the centromere region was used to improve the classification of several females in whom the copper uptake into cultured fibroblasts was either inconclusive or not available.     

The polymorphic human DNA sequence D8S8 assigned to 8ql3-21.1, close to the carbonic anhydrase gene cluster, by isotopic and non-isotopic in situ hybridization and by linkage analysis

Restriction fragment length polymorphism at the D8S8 locus is explained by the occurrence of at least two alternative alleles at two separate Taq I sites; Taql-A allele frequencies 0–73 and 0–27 and Taql-B allele frequencies 0–94 and 0–06. The D8S8 locus has been assigned to 8q 13–21.1, near to the carbonic anhydrase (CA) gene cluster, by in situ hybridization to metaphase chromosomes using both tritium and immunofluorescently labelled probes. Linkage analysis using the CEPH family DNA panel indicates a close genetic linkage between D8S8 and CA3 , with a lod score of + 7–80 at TH – 0.05 in males.     

Assignment of the human cardiac/slow skeletal muscle troponin C gene (TNNC1) between D3S3118 and GCT4B10 on the short arm of chromosome 3 by somatic cell hybrid analysis

We have determined the chromosomal location of the human cardiac/slow skeleletal muscle troponin C gene (TNNC1) to the short arm of chromosome 3 using somatic cell hybrids. PCR-based analysis of a 'monochromosomal' hybrid panel identified the presence of the TNNC1 gene on human chromosome 3 and subsequent analysis of the Genebridge4 radiation hybrid panel located the gene between D3S3118 (7·3cR) and GCT4B10 (4·2cR) with a lod score of >3·0.     

The gene responsible for Clouston hidrotic ectodermal dysplasia maps to the pericentromeric region of chromosome 13q

Hidrotic ectodermal dysplasia (HED), Clouston type, is an autosomal dominant skin disorder which is most common in the French-Canadian population and is characterized by hair defects, nail dystrophy and palmoplantar hyperkeratosis. Biophysical and biochemical studies conducted in HED suggested a molecular abnormality of keratins. We tested eight French-Canadian families segregating HED for linkage to microsatellite markers flanking the known keratin genes and were able to exclude linkage to these loci. Therefore, a genome-wide search for the HED gene was initiated. The first lod score above 3.00 was obtained with the marker D13S175 located in the pericentromeric region of chromosome 13q (Zmax = 8.12 at zero recombination). The cumulative lod scores were above 3.00 for six other markers in the region. A multipoint linkage analysis using the markers D13S175, D13S141 and D13S143 gave a maximum lod score of 11.12 at D13S141 with the one-lod- unit support interval spanning a 12.7 cM region which includes D13S175 and D13S141. Haplotype analysis allowed us to establish D13S143 as the telomeric flanking marker for the HED candidate region.      

Linkage studies in peripheral neurofibromatosis.

Peripheral neurofibromatosis (NF) is one of the most common major genetic disorders in man. Its chromosomal location is unknown and questions regarding such factors as genetic heterogeneity remain unanswered. We have ascertained and sampled several large multi-generation families for linkage studies including one family of 66 subjects, 28 of whom were affected with NF. Recombinant DNA studies of several restriction fragment length polymorphisms (RFLPs) including C3, ApoC2, pBam34 (D19S6], HAUP[APRT], pE40-1 [D11521], Hp[Hp2 alpha], LDR92, and LDR111 failed to show a significant linkage (Z [lod score] greater than or equal to 3.00) in this family. In addition, the results excluded areas of the genome around the marker loci (Z greater than or equal to - 2.00) as potential sites for linkage. The maximum Z obtained with the markers was for Hp at theta (maximum recombination fraction) = 0.20 and Z = 0.399. We are now in the process of screening additional RFLPs and families for linkage to NF.     

Multipoint mapping of adult onset polycystic kidney disease (PKD1) on chromosome 16.

Analysis of genetic linkage data in 33 adult onset polycystic kidney (ADPKD) families was carried out using probes for the D16S85, D16S84, and D16S94 loci. The data set of 33 families shows no evidence of genetic heterogeneity since one unlinked family was previously excluded. Two point linkage analysis showed maximum likelihood values of the recombination fraction of 0.07 for ADPKD and D16S85 (lod score 18.78), 0.02 for ADPKD and D16S84 (lod score 7.55), and 0.00 for ADPKD and D16S94 (lod score 6.73). Multipoint analysis showed a maximum likelihood order of tel-D16S85-0.06-D16S84-0.02-(PKD1, D16S94)-cen with a multipoint lod score of 32.16. Analysis of rare recombinants lying close to PKD1 gave results consistent with this order.     

Genetic linkage between Huntington''s disease and D4S10(G8) in Scottish families

Genetic linkage between Huntington's disease (HD) and polymorphic DNA markers at the D4S10 locus has been investigated in 16 Scottish families. A maximum lod score of 3.499 at a recombination fraction of 0.07 was found, with 95% confidence limits of 0.02 and 0.22. Only one obvious recombinant was detected, and the wide confidence limits probably reflect the large number of unaffected individuals whose risk could only be estimated empirically.     

Genomic cloning and localization by FISH and linkage analysis of the human gene encoding the primary subunit NMDAR1 (GRIN1) of the NMDA receptor channel

A cDNA clone of the NMDAR1 (isoform E) has been used to screen both lambda and eosmid genomic libraries. A genomic phage clone was identified and sequenced and was found to contain some of the 3′ coding regions of the GRIN1 gene. This clone was used to localize the gene using fluorescent in situ hybridization (FISH) to normal chromosomes, and also to a lymphoblastoid cell line containing a translocation involving chromosomes 9 and 15. FISH localized the gene to chromosome 9q34.3. The clone was used to screen a panel of genomic DNAs cut with 20 restriction enzymes. A VNTR sequence 5′ to the gene, which was polymorphic for a number of restriction enzymes, was detected. A PvuII fragment of the genomic clone was found to detect the VNTR on Southern hybridization. The polymorphic VNTR marker was mapped against chromosome 9q34 markers using linkage analysis in the CEPH families. The GRIN1 gene was linked to D9S7 with a maximum lod score of 20·09 at zero recombination fraction in males and 0·03 % recombination in females.     

A gene for autosomal recessive limb-girdle muscular dystrophy maps to chromosome 2p

The limb-girdle muscular dystrophies are a clinically and geneticallyheterogeneous group of disorders. We have ostudied two largeinbred families of different ethnic origin and excluded linkageto LGMD2 on chromosome 15q and SCARMD on chromosome 13. Proceedingto a genomic linkage search, we have now identified linkageto markers D2S134 and D2S136 on chromosome 2p (maximum lod score3.57 at zero recombination). The phenotype in the two familieswas similar, with onset in the pelvic girdle musculature inthe late teens and usually relatively slow progression. Thiswork Identifies a second locus for autosomal recessive limb-girdlemuscular dystrophy.     

Dyskeratosis congenita: three additional families show linkage to a locus in Xq28.

Dyskeratosis congenita (DC) is a rare inherited disorder with most families being of the X linked recessive type. We describe three families which show linkage to the marker DXS52 on Xq28. The combined maximum lod score was 2.00 at zero recombination. This is further evidence that the X linked DC gene is located at Xq28 and brings the reported maximum lod score for DC and DXS52 to 5.33 at zero recombination fraction, with a supporting recombination fraction interval of 0.00-0.10.     

Linkage in a family with X-linked Charcot-Marie-Tooth disease

The gene for the X-linked form of Charcot-Marie-Tooth disease (CMT Peroneal Muscular Atrophy, X-linked: McKusick No. 30280) has been shown in a single family to be linked to DXYS1 with a lod score of 4.55 at a recombination fraction of 0.03 and to PGK1 with a lod score of 3.34 at zero recombination. This is in agreement with previous work based on several families. Pooled data from this family and 7 previously reported families give a maximum lod score of 12.04 at theta max of 0.05 for linkage between CMTX and DXYS1 loci.     

REPORTS: Linkage of Pfeiffer syndrome to chromosome 8 centromere and evidence for genetic heterogeneity

Pfeiffer syndrome (PS) Is an autosomal dominant disorder characterizedby cranlosynostosis, mldfaclal hypoplasia, and broad thumbsand great toes. We examined 129 Individuals from 11 familieswith PS and performed linkage studies using microsatellite markersspanning the entire genome. Strongest support for linkage waswith DNA markers (D8S255, GATA8G08) from chromosome 8. Obligatecrossovers exclude close linkage to this region in six families,and there was significant evidence for genetic heterogeneity.A multipoint lod score of 7.15 was obtained In five families.The 11 cM Interval between D8S278 and D8S285 contains one genefor PS and also spans the centromere of chromosome 8.     

A gene responsible for cavernous malformations of the brain maps to chromosome 7q

Cavernous malformations of the brain are vascular lesions whichare present in up to 0.4% of all individuals and which are oftenaccompanied by seizures, migraine, hemorrhage and other neurologicproblems. Using linkage analysis and a set of short tandem repeatpolymorphisms, a gene responsible for cavernous malformationsin a large Hispanic kindred was mapped to the q11–q22region of chromosome 7. A maximum pairwise lod score of 4.2was obtained at zero recombination with marker PY5–18at locus D7S804. Lod scores in excess of 3.0 were obtained withfour additional markers closely linked to PY5–18. A broadchromosome 7q haplotype of 33 cM length on the sex average mapwas shared by all affected individuals indicating that the genelies between loci D7S502 and D7S479.     

Emery-Dreifuss muscular dystrophy: localisation to Xq27.3----qter confirmed by linkage to the factor VIII gene.

Two families with Emery-Dreifuss muscular dystrophy (EMD) have been studied with DNA markers mapping to Xq27.3----qter. No recombination was observed in 11 phase known meioses informative for the factor VIII gene (F8C) and eight phase known meioses informative for DXS15 (DX13), giving maximum lod scores of 3.50 and 2.50 respectively at a recombination fraction of zero. DXS52 (St14) showed one recombinant in 12 phase known meioses giving a maximum lod score of 2.62 at a recombination fraction of 0.07. These results map EMD to the distal end of the long arm of the X chromosome and are an important step in the development of tests for carrier detection and prenatal diagnosis.     

A closely linked DNA marker for facioscapulohumeral disease on chromosome 4q.

Close linkage of a hypervariable DNA probe on chromosome 4q (pH30, locus D4S139) has been found with the locus for facioscapulohumeral disease. Three recombinants were identified in a total of 140 meioses, giving a maximum lod score of 36.77 at a recombination fraction of 0.02. All but two of the families studied proved informative with this probe; all informative families showed evidence of linkage (except one family with a single scorable meiosis), making genetic heterogeneity unlikely from our data. The close linkage and highly informative nature of the probe will make it suitable for clinical application in presymptomatic and prenatal diagnosis. We have also confirmed loose linkage with the marker (Mfd22, locus D4S171) used to establish the initial assignment of the disorder to chromosome 4.