Recurrence risk due to germ line mosaicism: Duchenne and Becker muscular dystrophy.

The presence of multiple affected offspring from apparently non-carrier parents is caused by germ line mosaicism. Although germ line mosaicism has been reported for many diseases, figures for recurrence risks are known for only a few of them. In X-linked Duchenne and Becker muscular dystrophies (DMD/BMD), the recurrence risk for non-carrier females due to germ line mosaicism has been estimated to be between 14% and 20% (95% confidence interval 3-30) if the risk haplotype is transmitted. In this study, we have analyzed 318 DMD/BMD cases in which the detected mutation was de novo with the aim of obtaining a better estimate of the 'true' number of germ line mosaics and a more precise recurrence risk. This knowledge is essential for genetic counseling. Our data indicate a recurrence risk of 8.6% (4.8-12.2) if the risk haplotype is transmitted, but there is a remarkable difference between proximal (15.6%) (4.1-27.0) and distal (6.4%) (2.1-10.6) deletions. Overall, most mutations originated in the female. Deletions occur more often on the X chromosome of the maternal grandmother, whereas point mutations occur on the X chromosome of the maternal grandfather. In unhaplotyped de novo DMD/BMD families, the risk of recurrence of the mutation is 4.3%.

Duplications in the DMD gene.

The detection of duplications in Duchenne (DMD)/Becker Muscular Dystrophy (BMD) has long been a neglected issue. However, recent technological advancements have significantly simplified screening for such rearrangements. We report here the detection and analysis of 118 duplications in the DMD gene of DMD/BMD patients. In an unselected patient series the duplication frequency was 7%. In patients already screened for deletions and point mutations, duplications were detected in 87% of cases. There were four complex, noncontiguous rearrangements, with two also involving a partial triplication. In one of the few cases where RNA was analyzed, a seemingly contiguous duplication turned out to be a duplication/deletion case generating a transcript with an unexpected single-exon deletion and an initially undetected duplication. These findings indicate that for clinical diagnosis, duplications should be treated with special care, and without further analysis the reading frame rule should not be applied. As with deletions, duplications occur nonrandomly but with a dramatically different distribution. Duplication frequency is highest near the 5' end of the gene, with a duplication of exon 2 being the single most common duplication identified. Analysis of the extent of 11 exon 2 duplications revealed two intron 2 recombination hotspots. Sequencing four of the breakpoints showed that they did not arise from unequal sister chromatid exchange, but more likely from synthesis-dependent nonhomologous end joining. There appear to be fundamental differences therefore in the origin of deletions and duplications in the DMD gene.

Familial Paget's disease in The Netherlands: occurrence, identification of new mutations in the sequestosome 1 gene, and their clinical associations.

OBJECTIVE: To estimate the occurrence of familial Paget's disease of bone in The Netherlands, to examine the prevalence of mutations of the sequestosome 1 gene (SQSTM1) in identified families, and to assess potential genotype-phenotype associations. METHODS: We performed a case-control study of patients with Paget's disease and a mutation analysis of the SQSTM1 gene of index patients with familial disease and of the relatives of those with a mutation. Serum alkaline phosphatase (AP) activity was assessed, and bone scintigraphy was performed. RESULTS: Five percent of patients had at least 1 first-degree relative with the disease, compared with 0.5% of the controls (relative risk 10; 95% confidence interval 1.3-75.6). In 38.9% of patients with familial disease, heterozygous mutations in the SQSTM1 gene were identified. These were the previously described P392L mutation, which was present in 22.2% of patients, and 3 new mutations, S399P, G425R, M404T, 9 of which were present in 3 different families. All mutations were located in the ubiquitin-associated domain of the gene. There was a relationship between serum AP activity, as a marker of the disease, and the presence or absence of the G425R and P392L mutations, the subject's age, and the presence of Paget's disease. CONCLUSION: Our data provide further evidence of a causal role of SQSTM1 gene mutations in the pathogenesis of Paget's disease and allow the design of a strategy based on measurements of serum AP activity and age for investigating asymptomatic relatives of patients with familial Paget's disease of bone.

Somatic mosaicism of a point mutation in the dystrophin gene in a patient presenting with an asymmetrical muscle weakness and contractures.

We describe a patient with somatic mosaicism of a point mutation in the dystrophin gene causing benign muscular dystrophy with an unusual asymmetrical distribution of muscle weakness and contractures. To our knowledge this is the first patient with asymmetrical weakness and contractures in an ambulatory patient with a dystrophinopathy.

Dystrophin nonsense mutation induces different levels of exon 29 skipping and leads to variable phenotypes within one BMD family.

Within one X-linked muscular dystrophy family, different phenotypes for three males occurred: (1) a severely affected Becker patient with cardiomyopathy, (2) a mildly affected Becker patient, and (3) an apparently healthy male with elevated serum CK levels. In the muscle biopsy specimen of patient2 one out of four antibodies (NCL-DYS1) showed absence of dystrophin. The protein truncation test detected a truncated dystrophin for both muscle tissue and lymphocytes of this patient next to an additional near normal size fragment in muscle. Genomic sequence analysis revealed a nonsense mutation in exon 29 (4148C > T) of the dystrophin gene. Sequence analysis of the mRNA fragment of the larger peptide showed skipping of exon 29, restoring an open reading frame. Consequently, the epitope of the antibody NCL-DYS1 is mapped to exon 29. The variable clinical features of the three relatives from healthy to severely affected therefore seems to be related to the level of skipping of exon 29. This finding underscores the future potential of gene therapeutic strategies aimed at inducing exon skipping in Duchenne muscular dystrophy, to generate a much milder disease.

Sarcoglycanopathies in Dutch patients with autosomal recessive limb girdle muscular dystrophy.

Within a group of 76 sporadic/autosomal recessive limb girdle muscular dystrophy (LGMD) patients we tried to identify those with LGMD type 2C-E. Muscle biopsy specimens of 40 index patients, who had 22 affected sibs, were analyzed immuno-histochemically for the presence of three subunits: alpha-, beta-, and gamma-sarcoglycans. Abnormal sarcoglycan expression was established in eight patients, with six affected sibs. In one patient gamma-sarcoglycan was absent, and both alpha- and beta-sarcoglycans were reduced. In the remaining seven patients gamma-sarcoglycan was (slightly) reduced, and alpha- and beta-sarcoglycans were absent or reduced. By DNA sequencing mutations were detected in one of the three sarcoglycan genes in all eight cases. Three patients had mutations in the alpha-, three in the beta-, and two in the gamma-sarcoglycan gene. The patients with sarcoglycanopathy comprised the more severely affected cases (P=0.04). In conclusion, sarcoglycanopathy was identified in 23 % (14/62) of the autosomal recessive LGMD patients.

Diagnostic Notch3 sequence analysis in CADASIL: three new mutations in Dutch patients. Dutch CADASIL Research Group.

To confirm the clinical diagnosis in individual Dutch patients with cerebral autosomal dominant arteriopathy with subcortical infarcts and leukoencephalopathy (CADASIL), we performed direct sequence analysis of the abnormal gene, Notch3, in patients from 11 families without prior linkage analysis to chromosome 19. Eleven missense mutations involving the loss or gain of a cysteine residue were found, of which 3 are new. Exon 4 is a mutation hotspot (9 of 11 families). Notch3 sequence analysis of CADASIL patients in a diagnostic laboratory is a feasible procedure to confirm the clinical diagnosis in individual patients.

The clinical and molecular genetic approach to Duchenne and Becker muscular dystrophy: an updated protocol.

Detection of large rearrangements in the dystrophin gene in Duchenne and Becker muscular dystrophy is possible in about 65-70% of patients by Southern blotting or multiplex PCR. Subsequently, carrier detection is possible by assessing the intensity of relevant bands, but preferably by a non-quantitative test method. Detection of microlesions in Duchenne and Becker muscular dystrophy is currently under way. Single strand conformational analysis, heteroduplex analysis, and the protein truncation test are mostly used for this purpose. In this paper we review the available methods for detection of large and small mutations in patients and in carriers and propose a systematic approach for genetic analysis and genetic counselling of DMD and BMD families, including prenatal and preimplantation diagnosis.

Asymptomatic and late-onset ornithine transcarbamylase deficiency caused by a A208T mutation: clinical, biochemical and DNA analyses in a four-generation family.

We describe a 4-generation family in which a previously healthy 10-year-old boy died of late-onset ornithine transcarbamylase (OTC) deficiency. Pedigree analysis and allopurinol loading tests in female relatives were not informative. A missense mutation (A208T) in the OTC gene was detected in the deceased patient and in several clinically healthy male and female relatives, the oldest male being 97 years old. OTC deficiency was established in autopsy liver tissue of the propositus and liver biopsy samples of his sister, mother, and a maternal uncle. The males had 4% and 6% residual activity, respectively, the females 58% and 67%, respectively. The observed relation between the mutation and the decreased OTC activity in liver tissue of these subjects suggests that the mutation is a deleterious one. Late-onset, "mild" OTC deficiency can have a fatal or a favorable outcome. The disease can segregate undetected in families.

Asymptomatic and late-onset ornithine transcarbamylase (OTC) deficiency in males of a five-generation family, caused by an A208T mutation.

In a large five-generation Polish family, late-onset ornithine transcarbamylase (OTC) deficiency in males segregated with the missense mutation Ala208Thr (A208T), and all heterozygous females were asymptomatic. No other mutations were found in the coding sequences and intron-exon boundaries of the OTC gene. Surprisingly, the mutation originated from the great-grandfather of the index patient who died at age 59 of liver carcinoma. He never had dietary restrictions or hyperammonemic spells throughout life and appears to be the oldest male reported with OTC deficiency. The index patient had a severe OTC deficiency (3% of normal). Eight males died suddenly at ages 4 months to 23 years (average 14 years) after a foudroyant episode triggered by a common infection. The patients remained undiagnosed for 28 years because a metabolic defect was not considered to be the cause of the acute episodes. Recognition of the familial pattern of inheritance was initially unnoticed since the patients were admitted to eight different hospitals. DNA analysis predicted that two 'healthy' boys also had OTC deficiency, which was confirmed by abnormal results of allopurinol challenge tests. Initial suspicion of OTC deficiency in such families is complicated, since symptoms can develop at any age, or even remain absent. This obscures the typical pattern of X-linked inheritance in small families.

Point mutation screening for 16 exons of the dystrophin gene by multiplex single-strand conformation polymorphism analysis.

We have developed a rapid and nonradioactive method to screen for point mutations using the Pharmacia PhastSystem. In an SSCP analysis, we applied the two multiplex exon PCR kits, commonly used for the detection of deletions in Duchenne and Becker muscular dystrophy patients. The different exon bands in the multiplex SSCP pattern could be identified by running well-characterised deletion patients in this system. Two common polymorphisms were easily identifiable and are helpful in the haplotype analysis in families. Screening of 70 patients in which no gross rearrangement was detectable with the multiplex PCR and Southern blot, resulted in the identification of 6 patients with a band shift after SSCP analysis. Of these 6 band shifts, 5 were the result of a frame shift or termination mutation. The other band shift was found to be a rare polymorphism unlikely to be the cause of the patient's phenotype. Application of this technique enabled us to improve diagnosis in the families involved and will allow us to extend the search for point mutations in the remaining exons of the dystrophin gene.

First-trimester prenatal diagnosis in twin pregnancies.

Two twin pregnancies at risk for a sex-linked disorder are described. Both pregnancies were dichorionic. Transabdominal sampling was chosen for prenatal diagnosis. Molecular genetic techniques raised suspicion with regard to the accuracy of the samples in one case. Second-trimester amniocentesis confirmed the error. Selective feticide of the affected fetus was performed. When first-trimester prenatal diagnosis is offered in dichorionic twin pregnancies, confirmation through molecular genetic testing can confirm that villi have been obtained from different fetuses. All parties must be aware that additional invasive diagnostic procedures in the second trimester may be required in cases of doubt.

Different mosaicism frequencies for proximal and distal Duchenne muscular dystrophy (DMD) mutations indicate difference in etiology and recurrence risk.

In about 65% of the cases of Duchenne muscular dystrophy (DMD) a partial gene deletion or duplication in the dystrophin gene can be detected. These mutations are clustered at two hot spots: 30% at the hot spot in the proximal part of the gene and about 70% at a more distal hot spot. Unexpectedly we observed a higher frequency of proximal gene rearrangements among proved "germ line" mosaic cases. Of the 24 mosaic cases we are aware of, 19 (79%) have a proximal mutation, while only 5 (21%) have a distal mutation. This finding indicates that the mutations at the two hot spots in the dystrophin gene differ in origin. Independent support for the different mosaicism frequency was found by comparing the mutation spectra observed in isolated cases of DMD and familial cases of DMD. In a large two-center study of 473 patients from Brazil and the Netherlands, we detected a significant difference in the deletion distribution of isolated (proximal:distal ratio 1:3) and familial cases (ratio 1:1). We conclude from these data that proximal deletions most likely occur early in embryonic development, causing them to have a higher chance of becoming familial, while distal deletions occur later and have a higher chance of causing only isolated cases. Finally, our findings have important consequences for the calculation of recurrence-risk estimates according to the site of the deletion: a "proximal" new mutant has an increased recurrence risk of approximately 30%, and a "distal" new mutant has a decreased recurrence risk of approximately 4%.

Use of two multiplex (PCR) reactions as an initial delection screening method for DMD and BMD patients

O gene da distrofina, localizado em Xp21, é um loco gênico enorme, ocupando uma regiäo de mais de 2,3 Mb. Mutaçöes neste gene resultam na distrofia muscular Duchenne (DMD) ou distrofia muscular Becker (DMB). Cerca de 60% das mutaçöes säo deleçöes com um tamanho médio de 200 kb e säo detectáveis por análise de Southern blot usando sondas cDNA. Em 1988 Chamberlain et al. descreveram uma reaçäo multiplex para uma amplificaçäo simultânea de 9 exons do gene da distrofina, o que permitiu detectar cerca de 80% de todas as deleçöes. Recentemente Beggs et al. descreveram um conjunto adicional de primers para um teste multiplex de mais 9 exons. Usamos no Rio de Janeiro ambas as reaçöes multiplex no estudo de 27 pacientes DMD e 4 DMB. A síntese dos primers e o preparo dos kits foram realizados em Leiden. Com a reaçäo multiplex de Vhamberlain detectamos 10 deleçöes. A segunda reaçäo multiplex (Beggs) confirmou 7 dessas deleçöes e detectou mais 2 pacientes com deleçöes, uma para o exon 50 e outra para o exon 52. A análise Southern blot e a hibridizaçäo cDNA foram usadas para confirmar as deleçöes e determinar-lhes a extensäo. As sondas cDNA confirmaram as 12 deleçöes detectadas usando as duas reaçöes multiplex. Nossa experiência é a de que a abordagem multiplex para triagem inicial de deleçöes é um método bom e confiável

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.