Evaluation of universal immunohistochemical screening of sebaceous neoplasms in a service setting.

BACKGROUND: Muir-Torre syndrome (MTS) is a subtype of Lynch syndrome, which encompasses the combination of sebaceous skin tumours or keratoacanthomas and internal malignancy, due to mutations in DNA mismatch repair genes. Sebaceous neoplasms (SNs) may occur before other malignancies, and may lead to the diagnosis, which allows testing of other family members, cancer surveillance, risk-reducing surgery or prevention therapies. AIM: To evaluate the efficacy of universal immunohistochemistry (IHC) screening of SNs in a service setting. METHODS: Patients with SNs were ascertained by a regional clinical pathology service over a 3-year period. Results of tumour IHC, clinical genetics notes and germline genetic testing were retrospectively reviewed. RESULTS: In total, 62 patients presented with 71 SNs; 9 (15%) of these patients had previously diagnosed MTS. Tumour IHC was performed for 50 of the 53 remaining patients (94%); 26 (52%) had loss of staining of one or more mismatch repair proteins. Fifteen patients were referred to the Clinical Genetics department, and 10 patients underwent germline genetic testing. Two had a new diagnosis of MTS confirmed, with heterozygous pathogenic mutations detected in the MSH2 and PMS2 genes (diagnostic yield 20%). The PMS2 mutation was identified in a 57-year-old woman with a sebaceous adenoma and history of endometrial cancer; to our knowledge, this is the first time a PMS2 mutation has been reported in MTS. CONCLUSIONS: Universal IHC screening of SNs is an effective method to identify cases for further genetic evaluation. Rates of referral to clinical genetics were only moderate (58%). Increased awareness of MTS could help improve the rate of onward referral.

Mutations in RYR1 are a common cause of exertional myalgia and rhabdomyolysis.

Mutations in the skeletal muscle ryanodine receptor (RYR1) gene are a common cause of neuromuscular disease, ranging from various congenital myopathies to the malignant hyperthermia (MH) susceptibility trait without associated weakness. We sequenced RYR1 in 39 unrelated families with rhabdomyolysis and/or exertional myalgia, frequent presentations in the neuromuscular clinic that often remain unexplained despite extensive investigations. We identified 9 heterozygous RYR1 mutations/variants in 14 families, 5 of them (p.Lys1393Arg; p.Gly2434Arg; p.Thr4288_Ala4290dup; p.Ala4295Val; and p.Arg4737Gln) previously associated with MH. Index cases presented from 3 to 45 years with rhabdomyolysis, with or without exertional myalgia (n=12), or isolated exertional myalgia (n=2). Rhabdomyolysis was commonly triggered by exercise and heat and, less frequently, viral infections, alcohol and drugs. Most cases were normally strong and had no personal MH history. Inconsistent additional features included heat intolerance, and cold-induced muscle stiffness. Muscle biopsies showed mainly subtle changes. Familial RYR1 mutations were confirmed in relatives with similar or no symptoms. These findings suggest that RYR1 mutations may account for a substantial proportion of patients presenting with unexplained rhabdomyolysis and/or exertional myalgia. Associated clinico-pathological features may be subtle and require a high degree of suspicion. Additional family studies are paramount in order to identify potentially MH susceptible relatives.

Congenital myopathies--clinical features and frequency of individual subtypes diagnosed over a 5-year period in the United Kingdom.

The congenital myopathies are a group of inherited neuromuscular disorders mainly defined on the basis of characteristic histopathological features. We analysed 66 patients assessed at a single centre over a 5 year period. Of the 54 patients where muscle biopsy was available, 29 (54%) had a core myopathy (central core disease, multi-minicore disease), 9 (17%) had nemaline myopathy, 7 (13%) had myotubular/centronuclear myopathy, 2 (4%) had congenital fibre type disproportion, 6 (11%) had isolated type 1 predominance and 1 (2%) had a mixed core-rod myopathy. Of the 44 patients with a genetic diagnosis, RYR1 was mutated in 26 (59%), ACTA1 in 7 (16%), SEPN1 in 7 (16%), MTM1 in 2 (5%), NEB in 1 (2%) and TPM3 in 1 (2%). Clinically, 77% of patients older than 18 months could walk independently. 35% of all patients required ventilatory support and/or enteral feeding. Clinical course was stable or improved in 57/66 (86%) patients, whilst 4 (6%) got worse and 5 (8%) died. These findings indicate that core myopathies are the most common form of congenital myopathies and that more than half can be attributed to RYR1 mutations. The underlying genetic defect remains to be identified in 1/3 of congenital myopathies cases.

Mutations in MYH7 cause Multi-minicore Disease (MmD) with variable cardiac involvement.

Central Core Disease (CCD) and Multi-minicore Disease (MmD) (the "core myopathies") have been mainly associated with mutations in the skeletal muscle ryanodine receptor (RYR1) and the selenoprotein N (SEPN1) gene. A proportion of cases remain unresolved. Mutations in MYH7 encoding the beta myosin heavy chain protein have been implicated in cardiac and, less frequently, skeletal muscle disorders. Here we report four patients from two families with a histopathological diagnosis of MmD, presenting in childhood with slowly progressive muscle weakness, more proximal in Family 1 and more distal in Family 2, and variable degrees of cardiorespiratory impairment evolving later in life. There was also a strong family history of sudden death in the first family. Muscle biopsies obtained in early childhood showed multiple minicores as the most prominent feature. Sequencing of the MYH7 gene revealed heterozygous missense mutations, c.4399C>G; p.Leu1467Val (exon 32) in Family 1 and c.4763G>C; p.Arg1588Pro (exon 34) in Family 2. These findings suggest MYH7 mutations as another cause of a myopathy with multiple cores, in particular if associated with dominant inheritance and cardiac involvement. However, clinical features previously associated with this genetic background, namely a more distal distribution of weakness and an associated cardiomyopathy, may only evolve over time.

Relative frequency of congenital muscular dystrophy subtypes: analysis of the UK diagnostic service 2001-2008.

The Dubowitz Neuromuscular Centre is the UK National Commissioning Group referral centre for congenital muscular dystrophy (CMD). This retrospective review reports the diagnostic outcome of 214 UK patients referred to the centre for assessment of 'possible CMD' between 2001 and 2008 with a view to commenting on the variety of disorders seen and the relative frequency of CMD subtypes in this patient population. A genetic diagnosis was reached in 53 of 116 patients fulfilling a strict criteria for the diagnosis of CMD. Within this group the most common diagnoses were collagen VI related disorders (19%), dystroglycanopathy (12%) and merosin deficient congenital muscular dystrophy (10%). Among the patients referred as 'possible CMD' that did not meet our inclusion criteria, congenital myopathies and congenital myasthenic syndromes were the most common diagnoses. In this large study on CMD the diagnostic outcomes compared favourably with other CMD population studies, indicating the importance of an integrated clinical and pathological assessment of this group of patients.

X-linked myotubular myopathy due to a complex rearrangement involving a duplication of MTM1 exon 10.

X-linked myotubular myopathy is a predominantly severe congenital myopathy with central nuclei on muscle biopsy due to mutations in the MTM1 gene encoding myotubularin. We report a boy with typical features of X-linked myotubular myopathy. Sequencing of the MTM1 gene did not reveal any causative mutations. Subsequent MLPA analysis identified a duplication of MTM1 exon 10 both in the patient and his mother. Additional quantitative fluorescent PCR and long-range PCR revealed an additional large deletion (2536bp) within intron 10, 143bp downstream of exon 10, and confirmed the duplication of exon 10. Our findings suggest that complex rearrangements have to be considered in typically affected males with X-linked myotubular myopathy.

SEPN1-related myopathies: clinical course in a large cohort of patients.

OBJECTIVE: To assess the clinical course and genotype-phenotype correlations in patients with selenoprotein-related myopathy (SEPN1-RM) due to selenoprotein N1 gene (SEPN1) mutations for a retrospective cross-sectional study. METHODS: Forty-one patients aged 1-60 years were included. Clinical data including scoliosis, respiratory function, and growth measurements were collected by case note review. RESULTS: Mean age at onset was 2.7 years, ranging from birth to the second decade of life. All but 2 remained independently ambulant: one lost ambulation at age 5 years and another in his late 50s. The mean age of starting nocturnal noninvasive ventilation (NIV) was 13.9 years. One child required full-time NIV at the age of 1 year while in 2 cases NIV was started at 33 years. Two patients died from respiratory failure at the age of 10 and 22 years, respectively. The mean age at scoliosis onset was 10 years, in most cases preceded by rigidity of the spine. Fourteen patients had successful spinal surgery (mean age 13.9 years). Twenty-one were underweight; however, overt feeding difficulties were not a feature. CONCLUSIONS: This study describes the largest population affected by SEPN1-RM reported so far. Our findings show that the spectrum of severity is wider than previously reported. Respiratory insufficiency generally develops by 14 years but may occur as early as in infancy or not until the fourth decade. Motor abilities remain essentially static over time even in patients with early presentation. Most adult patients remain ambulant and fully employed.

Familial Alzheimer's disease and inherited prion disease in the UK are poorly ascertained.

AIMS: To ascertain the frequency and geographical distribution of patients diagnosed with known genetic causes of Alzheimer's disease (AD) and inherited prion disease (IPD) in the UK 2001-2005. By comparison with frequencies predicted from published population studies, to estimate the proportion of patients with these conditions who are being accurately diagnosed. METHODS: All the positive diagnostic test results (from both genetic testing centres) were identified for mutations in presenilin-1 (PSEN1), presenilin-2 (PSEN2), amyloid precursor protein (APP) and prion protein genes (PRNP) for patients resident in the UK in a 5 year period. The variation in the incidence of mutation detection between UK regions was assessed with census population data. Published studies of the genetic epidemiology of familial early onset AD (EOAD) were reviewed to produce estimates of the number of patients in the UK that should be detected. RESULTS: The rate of detection of EOAD and IPD varied very significantly and consistently between regions of the UK with low rates of detection in Northern and Western Britain (72% less detection in these regions compared with Central and Southeast Britain). The estimates from population studies further suggest a greater number of patients with EOAD than are diagnosed by genetic testing throughout the UK. CONCLUSIONS: It is likely that patients with EOAD and IPD are not being recognised and referred for testing. With the prospect of meaningful disease modifying therapeutics for these diseases, this study highlights an issue of relevance to neurologists and those planning for provision of National Health Services.

Congenital fibre type disproportion associated with mutations in the tropomyosin 3 (TPM3) gene mimicking congenital myasthenia.

Congenital myopathy with fibre type disproportion (CFTD) has been associated with mutations in ACTA1, SEPN1, RYR1 and TPM3 genes. We report the clinico-pathological and electrophysiological features of 2 unrelated cases with heterozygous TPM3 mutation. Case 1 is a 19-year-old lady who presented with motor delay in infancy, respiratory failure in early teens requiring non-invasive ventilation despite being ambulant, ptosis, axial more than proximal weakness and scoliosis. Case 2 is a 7-year-old boy with hypotonia, feeding difficulties, motor delay and scoliosis, also requiring non-invasive ventilation while ambulant. Muscle biopsies in both cases showed fibre type disproportion. Muscle MRI (Case 1) showed mild uniformly increased interstitial tissue in and around the muscles. Sequencing of TPM3 in case 1 revealed a previously described heterozygous c.503G > A(pArg168His) missense variant in exon 5 and a novel heterozygous missense mutation c.521A > C(pGlu174Ala), also in exon 5, in case 2. A mild abnormality in the single fibre EMG was documented on electrophysiology in both cases. These cases highlight the neuromuscular transmission defect in CFTD secondary to TPM3 mutations.

RYR1 mutations are a common cause of congenital myopathies with central nuclei.

OBJECTIVE: Centronuclear myopathy (CNM) is a rare congenital myopathy characterized by prominence of central nuclei on muscle biopsy. CNM has been associated with mutations in MTM1, DNM2, and BIN1 but many cases remain genetically unresolved. RYR1 encodes the principal sarcoplasmic reticulum calcium release channel and has been implicated in various congenital myopathies. We investigated whether RYR1 mutations cause CNM. METHODS: We sequenced the entire RYR1 coding sequence in 24 patients with a diagnosis of CNM from South Africa (n = 14) and Europe (n = 10) and identified mutations in 17 patients. The most common genotypes featured compound heterozygosity for RYR1 missense mutations and mutations resulting in reduced protein expression, including intronic splice site and frameshift mutations. RESULTS: The high incidence in South African patients (n = 12/14) in conjunction with recurrent RYR1 mutations associated with common haplotypes suggested the presence of founder effects. In addition to central nuclei, prominent histopathological findings included (often multiple) internalized nuclei and type 1 fiber predominance and hypotrophy with relative type 2 hypertrophy. Although cores were not typically seen on oxidative stains, electron microscopy revealed subtle abnormalities in most cases. External ophthalmoplegia, proximal weakness, and bulbar involvement were prominent clinical findings. INTERPRETATION: Our findings expand the range of RYR1-related phenotypes and suggest RYR1 mutations as a common cause of congenital myopathies with central nuclei. Corresponding to recent observations in X-linked CNM, these findings indicate disturbed assembly and/or malfunction of the excitation-contraction machinery as a key mechanism in CNM and related myopathies.

Intronic breakpoint definition and transcription analysis in DMD/BMD patients with deletion/duplication at the 5' mutation hot spot of the dystrophin gene.

Dystrophin mutations occurring at the 5' end of the gene frequently behave as exceptions to the "frame rule," their clinical severity being variable and often not related to the perturbation of the translation reading frame. The molecular mechanisms underlying the phenotypic variability of 5' dystrophin mutations have not been fully clarified. We have characterized the genomic breakpoints within introns 2, 6 and 7 and identified the splicing profiles in a cohort of DMD/BMD patients with deletion of dystrophin exons 3-7, 3-6 and duplication of exons 2-4. Our findings indicate that the occurrence of intronic cryptic promoter as well as corrective splicing events are unlikely to play a role in exons 3-7 deleted patients phenotypic variability. Our data suggest that re-initiation of translation could represent a major mechanism responsible for the production of a residual dystrophin in some patients with exons 3-7 deletion. Furthermore, we observed that the out-of-frame exon 2a is almost constantly spliced into a proportion of the dystrophin transcripts in the analysed patients. In the exons 2-4 duplicated DMD patient, producing both in-frame and out-of-frame transcripts, this splicing behaviour might represent a critical factor contributing to the severe phenotype. In conclusion, we suggest that multiple mechanisms may have a role in modulating the outcome of 5' dystrophin mutations, including recoding mechanisms and unusual splicing choices.

Development and implementation of a new rapid aneuploidy diagnostic service within the UK National Health Service and implications for the future of prenatal diagnosis.

BACKGROUND: Prenatal diagnosis for chromosome abnormality is routinely undertaken by full karyotype analysis of chromosomes from cultured cells; pregnant women must wait on average 13-14 days for their results. Autosomal trisomies, which account for around 80% of significant abnormalities, can be detected by quantitative fluorescence (QF) PCR. We report on the development and implementation of this technique as the first such routine service within a diagnostic department of the UK National Health Service (NHS). METHODS: We designed a "one-tube test" comprising four primer pairs for polymorphic tetranucleotide repeat sequences on chromosome 21, four primer pairs for sequences on chromosome 18, three primer pairs for sequences on chromosome 13, and one primer pair to identify the sex chromosomes. All prenatal samples received by our NHS diagnostic department between April, 2000, and April, 2001, were tested. After DNA extraction, PCR amplification was done and the products separated on a capillary-based genetic analyser; the results were interpreted with dedicated software. Follow-up karyotype analysis was done on all samples. FINDINGS: 1148 amniotic fluid samples, 188 chorionic villus samples, and 37 fetal tissue samples were tested; the amplification failure rate was zero with our current protocol. QF-PCR results were obtained and reported on 1314 (98%) of the prenatal samples; the remaining 22 (2%) were uninformative because of maternal-cell contamination. One case of mosaicism in a chorionic villus sample, and two cases indicating somatic expansion of a tetranucleotide repeat were found. No false positive or false negative results were obtained. The mean reporting time for the last 4 months of data collection was 1.25 working days. INTERPRETATION: QF-PCR aneuploidy testing is an efficient and accurate technique for the detection of autosomal trisomies in prenatal samples. Implementation of this service has led to the rapid diagnosis of abnormalities and early reassurance for women with normal results.

Six unaffected livebirths following preimplantation diagnosis for spinal muscular atrophy.

Spinal muscular atrophy (SMA) is a severe neurodegenerative autosomal recessive disorder, second only in frequency to cystic fibrosis. In its most severe form, SMA type I (Werdnig-Hoffman), death invariably ensues before age 2 years from respiratory failure or infection. Around 98% of clinical cases of SMA are caused by the homozygous absence of a region of exons 7 and 8 of the telomeric copy of the SMN gene (SMN1) on chromosome 5. We have developed a novel means of preimplantation diagnosis of SMA using a nested polymerase chain reaction (PCR) amplification of exon 7 of SMN, followed by a HinfI restriction digest of the PCR product enabling the important SMN1 gene to be distinguished from the centromeric SMN2 gene which has no clinical phenotype. This method was designed to reduce the likelihood of misdiagnosis. Five couples were treated using this method. Four proceeded to embryo transfer which resulted in six liveborns (one singleton, one twin and one triplet), all free of SMA. Embryo transfer was not performed in one cycle because of PCR contamination.

Comparative mutation detection screening of the type VII collagen gene (COL7A1) using the protein truncation test, fluorescent chemical cleavage of mismatch, and conformation sensitive gel electrophoresis.

Mutations in the type VII collagen gene, COL7A1, give rise to the blistering skin disease, dystrophic epidermolysis bullosa. We have developed two new mutation detection strategies for the screening of COL7A1 mutations in patients with dystrophic epidermolysis bullosa and compared them with an established protocol using conformational sensitive gel electrophoresis. The first strategy consisted of an RNA based protein truncation test that amplified the entire coding region in only four overlapping nested reverse transcriptase-polymerase chain reaction assays. These fragments were transcribed and translated in vitro and analyzed using sodium dodecyl sulfate-polyacrylamide gel electrophoresis. We have used the protein truncation test procedure to characterize 15 truncating mutations in 13 patients with severe recessive dystrophic epidermolysis bullosa yielding a detection sensitivity of 58%. The second strategy was a DNA-based fluorescent chemical cleavage of mismatch (fl-CCM) procedure that amplified the COL7A1 gene in 21 polymerase chain reaction assays. Mismatches, formed between patient and control DNA, were identified using chemical modification and cleavage of the DNA. We have compared fl-CCM with conformational sensitive gel electrophoresis by screening a total of 50 dominant and recessive dystrophic epidermolysis bullosa patients. The detection sensitivity for fl-CCM was 81% compared with 75% for conformational sensitive gel electrophoresis (p = 0.37 chi2-test). Using a combination of the three techniques we have screened 93 dystrophic epidermolysis bullosa patients yielding an overall sensitivity of 87%, detecting 79 different mutations, 57 of which have not been reported previously. Comparing all three approaches, we believe that no single method is consistently better than the others, but that the fl-CCM procedure is a sensitive, semiautomated, high throughput system that can be recommended for COL7A1 mutation detection.

Identification and quantification of somatic mosaicism for a point mutation in a Duchenne muscular dystrophy family.

Relatively few point mutations have been found in the dystrophin gene and of these only two have been associated with mosaicism. A single base insertion has been identified and quantified in a mother of two sons affected with Duchenne muscular dystrophy. It has been determined that she is a somatic mosaic with the mutation present in 25% of lymphocyte DNA. Further tissue lineages have been tested and the time at which the mutation arose was determined to be before the cellular differentiation into the bilaminar disc at approximately eight days after fertilisation. We suggest that the incidence of mosaicism for dystrophin point mutations may be higher than current data suggest.

Correlation of SMNt and SMNc gene copy number with age of onset and survival in spinal muscular atrophy.

Childhood-onset autosomal recessive spinal muscular atrophy (SMA) is associated with absence of the telomeric survival motor neuron gene (SMNt) in most patients, and deletion of the neuronal apoptosis inhibitory protein (NAIP) gene in the majority of severely affected patients. Analysis of SMNt has been complicated by the existence of a centromeric copy, SMNc, which is almost identical to SMNt but which can be distinguished from it by restriction enzyme analysis. In this study 143 SMA patients have been genotyped for the presence or absence of the SMNt, SMNc and NAIP genes, and the data correlated with quantifiable clinical variables. Although a significant correlation was observed between the presence or absence of the NAIP gene and the severity of the clinical phenotype in SMA patients generally, there was no difference in age of onset or survival in type I patients with the NAIP+ or NAIP- genotype. Fluorimetric PCR analysis of SMNc gene dosage in 57 patients homozygous for the absence of the SMNt gene but in whom the NAIP gene was present showed a highly significant correlation between SMNc copy number and SMA subtype, and between SMNc copy number and both age of onset and length of survival. The data provide strong statistical support for the emerging consensus that the clinical phenotype in SMA is directed primarily by the level of functional SMN protein. The lower SMNc copy number in type I patients in whom the NAIP gene is present suggests that the SMNt gene is removed by deletion in the majority of such patients, rather than by gene conversion as is the case in SMA types II and III.

Dystrophin point mutation screening using a multiplexed protein truncation test.

We report here the first use of a multiplexed protein truncation test for the high throughput screening of dystrophin point mutations. We have developed a substantially more robust and efficient procedure incorporating large savings in cost which uses muscle biopsy or lymphocyte total RNA as the template. The entire dystrophin open reading frame is screened in only five overlapping fragments using a long RT-PCR strategy to amplify dystrophin cDNA in excess of 3.7 kb. These five fragments are uniquely transcribed and translated in vitro in a single multiplexed reaction containing magnesium ions to reduce nonspecific internal initiation of translation. We have used this system to analyze mutations in 11 Duchenne muscular dystrophy patients (10 unrelated) with previously uncharacterized mutations. A single truncating mutation was identified in all patients, which was confirmed at the genomic level. Multiplex PTT provides the most efficient method for point mutation screening in this large gene and has potential applications to several disease genes with a significant proportion of truncating mutations.

Prenatal diagnosis of Duchenne and Becker muscular dystrophy.

Prenatal diagnosis of Duchenne and Becker muscular dystrophy is performed as a routine procedure in many laboratories around the world, using numerous molecular genetic techniques. Rather than discussing methods that are commonly in use, this review concentrates on some of the methods that are less widely available. This includes techniques than can be applied to routine diagnosis, to difficult cases where DNA analysis is unhelpful, and alternatives to standard methods of prenatal diagnosis.

Accurate diagnosis of carriers of deletions and duplications in Duchenne/Becker muscular dystrophy by fluorescent dosage analysis.

We have developed a semiautomated approach to amplify 25 exons of the dystrophin gene using two fluorescent multiplex PCR assays which detect over 98% of reported deletions and 90% of duplications causing Duchenne/Becker muscular dystrophy. The 5' multiplex detects 11 exons from the proximal deletion hotspot of the gene while the 3' multiplex detects 14 exons from the central deletion hotspot. The PCR products are accurately sized and quantified by a fluorescent DNA sequencer after only 18 cycles of amplification. The amount of product amplified from each exon in a multiplex is divided by that from each of the other exons, and this ratio is compared with those from control samples to obtain a series of dosage quotients (DQ), from which the copy number of each exon is determined. No overlap was observed between the DQ values obtained from single and double copy loci. The assays can be used to screen both affected males and at risk female relatives for a mutation. The method has been evaluated as a female carrier test by conducting a blind trial on 150 coded samples. Sixty-three deletion carriers, two duplication carriers, and 84 normal female controls were all correctly identified, showing that carrier diagnosis is possible even in families where the nature of the mutation is unknown. Additionally the analysis showed a non-pathogenic duplication involving the muscle specific promoter and exon 1. Together these two multiplex assays detect over 70% of all mutations in the dystrophin gene, greatly simplifying and partly automating molecular diagnosis in Duchenne and Becker muscular dystrophy.

Deletions in the 5' region of dystrophin and resulting phenotypes.

Deletions in the dystrophin gene give rise to both Duchenne and Becker muscular dystrophies. Good correlation is generally found between the severity of the phenotype and the effect of the deletion on the reading frame: deletions that disrupt the reading frame result in a severe phenotype, while in frame deletions are associated with a milder disease course. Rare exceptions to this rule, mainly owing to frameshift mutations in the 5' region of the gene (in particular deletions involving exons 3 to 7) which are associated with a milder than expected phenotype, have been reported previously. In order to characterise better the relationship between genotype and phenotype as a result of mutations arising in the 5' region of the gene, we have studied a large cohort of patients with small in frame and out of frame deletions in the first 13 exons of the dystrophin gene. Fifty-five patients with a deletion in this area were identified; approximately one third of them had a phenotype different from that theoretically expected. Patients were divided into two groups: (1) patients with a severe clinical phenotype despite the presence of a small, in frame deletion and (2) patients with a mild phenotype and an out of frame deletion. Noticeable examples observed in the first group were Duchenne boys with a deletion of exon 5, of exon 3, and of exons 3-13. In the second group we observed several patients with an intermediate or Becker phenotype and out of frame deletions involving not only the usual exons 3-7 but also 5-7 and 3-6. These data indicate that a high proportion of patients with a deletion in the 5' end of the gene have a phenotype that is not predictable on the basis of the effect of the deletion on the reading frame. The N-terminus of dystrophin has at least one actin binding domain that might be affected by the small, in frame deletions in this area. The effect of the in frame deletions of exon 3, 5, and 3-13 on this domain might account for the severe phenotype observed in these patients. Other mechanisms, such as unexpected effect of the deletion on splicing behaviour, might, however, also be implicated in determining the phenotype outcome.