H Scheffer

Revertant Mosaicism in Epidermolysis Bullosa Caused by Mitotic Gene Conversion

Mitotic gene conversion acting as reverse mutation has not been previously demonstrated in human. We report here that the revertant mosaicism of a compound heterozygous proband with an autosomal recessive genodermatosis, generalized atrophic benign epidermolysis bullosa, is caused by mitotic gene conversion of one of the two mutated COL17A1 alleles. Specifically, the maternal allele surrounding the mutation site on COL17A1 (1706delA) showed reversion of the mutation and loss of heterozygosity along a tract of at least 381 bp in revertant keratinocytes derived from clinically unaffected skin patches; the paternal mutation (R1226X) remained present in all cell samples. Revertant mosaicism represents a way of natural gene therapy.

Association between angiotensin-converting-enzyme gene polymorphism and failure of renoprotective therapy

BACKGROUNDnPolymorphism in the gene for angiotensin-converting enzyme (ACE), especially the DD genotype, is associated with risk for cardiovascular disease. Glomerulosclerosis has similarities to atherosclerosis, and we looked at ACE gene polymorphism in patients with kidney disease who were in a trial of long-term therapy with an ACE inhibitor or a beta-blocker.nnnMETHODSn81 patients with non-diabetic renal disease had been entered into a randomised comparison of oral atenolol or enalapril to prevent progressive decline in renal function. The dose was titrated to a goal diastolic blood pressure of 10 mm Hg below baseline and/or below 95 mm Hg. The mean (SE) age was 50 (1) years, and the group included 49 men. Their renal function had been monitored over 3-4 years. We have looked at their ACE genotype, which we assessed with PCR.nnnFINDINGSn27 patients had the II genotype, 37 were ID, and 17 were DD. 11 patients were lost to follow-up over 1-3 years. The decline of glomerular filtration rate over the years was significantly steeper in the DD group than in the ID and the II groups (p = 0.02; means -3.79, -1.37, and -1.12 mL/min per year, respectively). The DD patients treated with enalapril fared as equally a bad course as the DD patients treated with atenolol. Neither drug lowered the degree of proteinuria in the DD group.nnnINTERPRETATIONnOur data show that patients with the DD genotype are resistant to commonly advocated renoprotective therapy.

PARENTAL ORIGIN AND GERMLINE MOSAICISM OF DELETIONS AND DUPLICATIONS OF THE DYSTROPHIN GENE - A EUROPEAN STUDY

SummaryKnowledge about the parental origin of new mutations and the occurrence of germline mosaicism is important for estimating recurrence risks in Duchenne (DMD) and Becker muscular dystrophy (BMD). However, there are problems in resolving these issues partly because not all mutations can as yet be directly detected, and additionally because genetic ratios are very sensitive to ascertainment bias. In the present study, therefore, analysis was restricted to currently detectable mutations (deletions and duplications) in particular types of families which tend to be rare. In order to obtain sufficient data we pooled results from 25 European centers. In mothers of affected patients who were the first in their family with a dystrophin gene deletion or duplication, the ratio between the paternal and the maternal origin of this new mutation was 32:49 (binomial test P = 0.075) for DMD. In five BMD families the ratio between paternal and maternal origin of new mutations was 3∶2. Recurrence risk because of maternal germline mosaicism was studied in sisters or subsequent sibs of isolated cases with an apparently new detectable mutation. In 12 out of 59 (0.20; 95% CI 0.10–0.31) transmissions of the risk haplotype the DMD mutation was transmitted as well. No recurrences were found in nine BMD families.

Glycogen storage disease type Ia: recent experience with mutation analysis, a summary of mutations reported in the literature and a newly developed diagnostic flowchart

Abstract We studied the glucose-6-phosphatase (G6Pase) gene of 30 unrelated glycogen storage disease type Ia (GSD Ia) patients using single strand conformational polymorphism (SSCP) prior to automated sequencing of exons revealing an aberrant SSCP pattern. In all patients we could identify mutations on both alleles of the G6Pase gene, indicating that this method is a reliable procedure. A total of 14 different mutations were identified. R83C (16/60), 158delC (12/60), Q347X (7/60), R170X (6/60) and ΔF327 (4/60) were found most frequently. Nine other mutations accounted for the other 15 mutant alleles. Two DNA-based prenatal diagnoses were performed successfully. At present, 56 mutations in the G6Pase gene have been reported in 300 unrelated GSD Ia patients and an overview of these mutations is presented. Evidence for a clear genotype-phenotype correlation could be established neither from our data nor from those in the literature. With increased knowledge about the genetic basis of GSD Ia and GSD Ib and the high detection rate of mutations, it is our opinion that the diagnoses GSD Ia and GSD Ib can usually be based on clinical and biochemical abnormalities combined with mutation analysis instead of enzyme assays in liver tissue obtained by biopsy. A newly developed flowchart for the diagnosis of GSD I is presented.nConclusion Increased knowledge of the genetic basis of glycogen storage disease type I provides a DNA-based diagnosis, prenatal DNA-based diagnosis in chorionic villus samples and carrier detection.n

Mutation frequency of cystic fibrosis transmembrane regulator is not increased in oligozoospermic male candidates for intracytoplasmic sperm injection

OBJECTIVEnTo examine the frequency of anomalies of the vas deferens and the frequency of mutations of the cystic fibrosis transmembrane regulator (CFTR) gene in male candidates for intracytoplasmic sperm injection (ICSI) who had severe oligoasthenoteratozoospermia.nnnDESIGNnThe clinical data for male candidates for ICSI were studied. The three most frequent cystic fibrosis (CF)-causing CFTR mutations in the Dutch population (deltaF508, A455E, and G542X) and the three most frequent CFTR mutations potentially causing congenital bilateral absence of the vas deferens (CBAVD) in the Dutch population (deltaF508, R117H, and IVS8-5T) were analyzed. Delta I507 is also detected by the deltaF508 test. Samples of DNA from patients identified as CFTR mutation carriers were subjected to denaturing gradient gel electrophoresis analysis with use of a two-dimensional electrophoretic technique.nnnSETTINGnUniversity-based center for reproductive medicine and clinical genetics.nnnPATIENT(S)nMale candidates for ICSI who had oligoasthenoteratozoospermia and no history of operative sterilization and refertilization. Males with a chromosomal aberration or a Y-chromosome microdeletion were excluded.nnnINTERVENTION(S)nSemen and blood samples were collected from the patients at their first visit to the clinic.nnnMAIN OUTCOME MEASURE(S)nFrequency of anomalies of the vas deferens and frequency of mutations of the CFTR gene in male candidates for ICSI who had oligoasthenoteratozoospermia.nnnRESULT(S)nNone of the patients had abnormalities of the vas deferens at physical examination. In 4 of the 150 chromosomes (75 patients), a CFTR mutation was found, yielding a CFTR mutation frequency of 2.7% (95% confidence interval, 1.0-6.7%). None of the patients had two CFTR mutations.nnnCONCLUSION(S)nThe frequency of congenital abnormalities of the vas deferens in patients with oligoasthenoteratozoospermia is low. The frequencies of the CFTR mutations identified in this cohort did not differ significantly from the frequencies found in the normal Dutch population.

Maternal uniparental disomy for chromosome 14 in a boy with a normal karyotype

We report on a boy with a maternal uniparental disomy for chromosome 14 (UPD(14)). At 7 years of age he was referred to us by the paediatrician because of symptoms of Prader-Willi syndrome (PWS). He showed short stature, obesity, mild developmental delay, cryptorchidism, and some mild dysmorphic features. The history further indicated intrauterine growth retardation at the end of the pregnancy. His mother was 44 years of age at the time of his birth. After birth he showed hypotonia with poor sucking, for which gavage feeding was needed. Motor development was delayed. After 1 year he became obese despite a normal appetite. Recurrent middle ear infections, a high pain threshold, and a great skill with jigsaw puzzles were reported. There were no behavioural problems or sleep disturbance. Chromosomal analysis was normal (46,XY). DNA analysis for Prader-Willi syndrome showed no abnormalities. Two years later he was re-examined because we thought his features fitted the PWS-like phenotype associated with maternal UPD(14). At that time precocious puberty was evident. DNA analysis showed maternal heterodisomy for chromosome 14. In all the previously described 11 cases with maternal UPD(14), a Robertsonian translocation involving chromosome 14 was detected cytogenetically before DNA analysis. This is the first report of diagnosis of maternal UPD(14) based on clinical features. This finding underlines the importance of DNA analysis for maternal UPD(14) in patients with a similar PWS-like phenotype even without previous identification of a Robertsonian translocation involving chromosome 14.

Three novel KCNA1 mutations in episodic ataxia type I families

Hereditary paroxysmal ataxia, or episodic ataxia (EA), is a rare, genetically heterogeneous neurological disorder characterized by attacks of generalized ataxia. By direct sequence analysis, a different missense mutation of the potassium channel gene (KCNA1) has been identified in three families with EA.

242 Breakpoints in the 200-kb Deletion-prone P20-region of the Dmd Gene Are Widely Spread

Using whole cosmids as probes, we have mapped 242 DMD/BMD deletion breakpoints located in the major deletion hot spot of the DMD gene. Of these, 113 breakpoints were mapped more precisely to individual restriction enzyme fragments in the distal 80 kb of the 170-kb intron 44. An additional 12 breakpoints are distributed over the entire region, with no significant local variation in frequency. Furthermore, deletion sizes vary and are not influenced by the positions of the breakpoints. This argues against a predominant role of one or a few specific sequences in causing frequent rearrangements. It suggests that structural characteristics or a more widespread recombinogenic sequence makes this region so susceptible to deletion. Our study revealed several RFLPs, one of which is a 300-bp insertion/deletion polymorphism. Abnormally migrating junction fragments are found in 81% of the precisely mapped deletions and are highly valuable in the diagnosis of carrier females.

Validation of the determination of Delta F508 mutations of the cystic fibrosis gene in over 11000 mouthwashes

Mouthwashes can be used as a DNA resource for mutation detection and, because collection and DNA isolation is simple and cheap, they could in particular, be used for large numbers of samples. To determine the failure rate (the proportion of mouth samples in which no PCR product was obtained) and the specificity of buccal epithelial cell mutation detection in large numbers of samples, we collected mouthwashes and blood samples from 11413 blood donors and tested the mouthwashes for the ΔF508 mutation, which has an estimated frequency of 75% among cystic fibrosis chromosomes in The Netherlands. Blood samples were tested for the ΔF508 mutations only if the mutation was identified in the mouthwash or in the case of a failure to obtain PCR products. The sensitivity of the test was determined in mouthwashes of 75 ΔF508 carriers known from earlier family studies. These samples were offered blindly between the mouthwashes of the blood donors. Both specificity and sensitivity of the mouthwash procedure were 100%. The overall failure rate was 5.6%. This large figure was caused mainly by insufficient rinsing of the mouth in one particular blood bank. Exclusion of the results of this blood bank reduced the failure rate to 1.8%. Our results also confirm that for a large number of samples the mouthwash procedure is suitable for mutation detection and, with proper instructions, can be used in community screening.

Comprehensive and accurate mutation scanning of the CFTR gene by two-dimensional DNA electrophoresis

The large number of possible disease‐causing mutations in the 27 exons of the cystic fibrosis transmembrane conductance regulator (CFTR) gene has severely limited direct diagnosis of cystic fibrosis (CF) patients and carriers by mutation detection. Here we show that in principle testing for mutations in the CFTR gene can be both substantially facilitated and made virtually complete, by two‐dimensional DNA electrophoretic separation of polymerase chain reaction (PCR) amplified exons on the basis of size and basepair sequence in denaturing gradient gels. Under a single optimized set of conditions we were able to obtain a pattern of spots representing all 27 exons of the CFTR gene and to readily detect 17 out of 17 identified sequence variations in 9 different exons in DNA from 11 CF patients and carriers. Our results demonstrate the potential of 2‐dimensional DNA electrophoresis for comprehensive mutation analysis of the CFTR gene. The approach serves as a model for comprehensive diagnosis of the many other large disease genes for which a variety of mutations have also which been reported.