A missense mutation in the dystrophin gene in a Duchenne muscular dystrophy patient.

About two thirds of Duchenne muscular dystrophy (DMD) patients have either gene deletions or duplications. The other DMD cases are most likely the result of point mutations that cannot be easily identified by current strategies. Utilizing a heteroduplex technique and direct sequencing of amplified products, we screened our nondeletion/duplication DMD population for point mutations. We now describe what we believe to be the first dystrophin missense mutation in a DMD patient. The mutation results in the substitution of an evolutionarily conserved leucine to arginine in the actin-binding domain. The patient makes a dystrophin protein which is properly localized and is present at a higher level than is observed in DMD patients. This suggests that an intact actin-binding domain is necessary for protein stability and essential for function.

Comparison of cytogenetic and molecular genetic detection of t(8;21) and inv(16) in a prospective series of adults with de novo acute myeloid leukemia: a Cancer and Leukemia Group B Study.

PURPOSE: To prospectively compare cytogenetics and reverse transcriptase-polymerase chain reaction (RT-PCR) for detection of t(8;21)(q22;q22) and inv(16)(p13q22)/t(16;16)(p13;q22), aberrations characteristic of core-binding factor (CBF) acute myeloid leukemia (AML), in 284 adults newly diagnosed with primary AML. PATIENTS AND METHODS: Cytogenetic analyses were performed at local laboratories, with results reviewed centrally. RT-PCR for AML1/ETO and CBFbeta/MYH11 was performed centrally. RESULTS: CBF AML was ultimately identified in 48 patients: 21 had t(8;21) or its variant and AML1/ETO, and 27 had inv(16)/t(16;16), CBFbeta/MYH11, or both. Initial cytogenetic and RT-PCR analyses correctly classified 95.7% and 96.1% of patients, respectively (P =.83). Initial cytogenetic results were considered to be false-negative in three AML1/ETO-positive patients with unique variants of t(8;21), and in three CBFbeta/MYH11-positive patients with, respectively, an isolated +22; del(16)(q22),+22; and a normal karyotype. The latter three patients were later confirmed to have inv(16)/t(16;16) cytogenetically. Only one of 124 patients reported initially as cytogenetically normal was ultimately RT-PCR-positive. There was no false-positive cytogenetic result. Initial RT-PCR was falsely negative in two patients with inv(16) and falsely positive for AML1/ETO in two and for CBFbeta/MYH11 in another two patients. Two patients with del(16)(q22) were found to be CBFbeta/MYH11-negative. M4Eo marrow morphology was a good predictor of the presence of inv(16)/t(16;16). CONCLUSION: Patients with t(8;21) or inv(16) can be successfully identified in prospective multi-institutional clinical trials. Both cytogenetics and RT-PCR detect most such patients, although each method has limitations. RT-PCR is required when the cytogenetic study fails; it is also required to determine whether patients with suspected variants of t(8;21), del(16)(q22), or +22 represent CBF AML. RT-PCR should not replace cytogenetics and should not be used as the only diagnostic test for detection of CBF AML because of the possibility of obtaining false-positive or false-negative results.

Over-representation of PPARgamma sequence variants in sporadic cases of glioblastoma multiforme: preliminary evidence for common low penetrance modifiers for brain tumour risk in the general population.

PPARgamma, the gamma isoform of a family of peroxisome proliferator activated receptors, plays a key role in adipocyte differentiation. Recently, its broad expression in multiple tissues and several epithelial cancers has been shown. Further, somatic loss of function mutations in PPARgamma have been found in primary colorectal carcinomas. We sought to determine if somatic high penetrance mutations in this gene might also play a role in glioblastoma multiforme (GBM). We also examined this gene to determine if common low penetrance polymorphic alleles might lend low level susceptibility to GBM in the general population. No somatic high penetrance mutations were detected in 96 sporadic GBMs. However, polymorphic alleles at codons 12 and 449 were significantly over-represented among the 27 unrelated American patients with sporadic GBM compared to 80 race matched controls. While nine (33%) were heterozygous for the P12A variant, c.34C/G (cytosine to guanine change at nucleotide 34), 12 (15%) controls were heterozygous for P12A (p<0.05). Similarly, 13 of 26 (50%) glioblastoma patients compared to 10 of 80 (12%) normal controls were found to have the heterozygous H449H polymorphism (p<0.001). The over-representation of H449H in glioblastoma patients was confirmed with a second validation set of American patients. When both American series were combined, polymorphic H449H was over-represented among cases versus controls (p<0.001) and there was a similar trend (p=0.07) for P12A. The precise mechanism for this association is unknown but these PPARgamma polymorphisms may be acting in a low penetrance predisposing manner. However, these associations were not found in a German population, possibly arguing that if these variants are in linkage disequilibrium with a third locus, then this effect is relatively new, after the settlement of the American colonies.

Polymorphisms in a pseudogene highly homologous to PMS2.

PMS2 is one of a complex of genes encoding DNA repair proteins that includes MSH2, MLH1, MSH6 and MSH3. Mutation of any of these DNA mismatch repair genes leads to impairment of DNA repair and can lead to tumorigenesis. Germline mutation of PMS2 has been reported as a rare cause of hereditary nonpolyposis colorectal cancer (HNPCC) and Turcot's syndrome. The PMS2 gene is located on chromosome 7p22 and consists of 15 exons. Within exon 11 of PMS2 is a coding repeat of eight adenosines. This study reports on the finding of a nonexpressed pseudogene that is highly homologous to the PMS2 gene in this region. The pseudogene is polymorphic for two alterations in the repeat region: a 3 bp delAAA at a site corresponding to nucleotide 1231 in PMS2; and an AA-->GG change at nucleotide 1238. Due to the high homology in both intronic and exonic sequences, polymorphisms in this pseudogene could be mistaken for mutations in the PMS2 gene and erroneously thought to be a cause of HNPCC and/or Turcot's syndrome.

Kennedy's disease: a clinicopathologic correlation with mutations in the androgen receptor gene.

We confirmed a mutation of the androgen receptor gene as the cause for Kennedy's disease, also called "X-linked recessive spinal and bulbar muscular atrophy" or "bulbospinal neuronopathy." The mutation is characterized by an increased size of a polymorphic tandem CAG repeat within the first exon of the gene. The study population consisted of 17 patients from seven families (five distinct kinships and two isolated cases). Two patients were as yet asymptomatic and had normal examinations. Four carrier females showed the mutant as well as the normal allele; none showed clinical features of Kennedy's disease. There was no large expansion of the mutation observed in three generations of one family. Phenotypic expression between and within families was variable and not related to the size of the mutation. This contrasts with the gene mutations found in myotonic dystrophy and fragile X syndrome, where increased severity of disease correlates with the number of tandem triplet repeats. The findings reported here appear to explain the failure to find genetic anticipation in Kennedy's disease. The DNA test for Kennedy's disease can now be used for definitive diagnosis and carrier detection. In addition, mutation analysis allows early detection, which has implications for potential treatment.

Diagnosis of Duchenne dystrophy by enhanced detection of small mutations.

OBJECTIVE: To determine whether detection of small mutations of the dystrophin gene can be increased using an enhanced method of single-strand conformation polymorphism analysis. BACKGROUND: Usual methods of DNA analysis for Duchenne dystrophy cannot identify mutations in one-third of cases. Muscle biopsy, with its inherent risks and added liability for patients with Duchenne dystrophy, becomes the sole method of diagnosis. Even with a tissue diagnosis of dystrophin deficiency, many families are excluded from carrier detection and prenatal diagnosis. METHODS: Genomic DNA from a cohort of 93 patients with Duchenne dystrophy without identifiable gene mutations was screened for mutations. In each case, 22 kilobases of genomic DNA were scanned, including all 79 exons of the dystrophin gene, adjacent intronic regions, and six alternative exons 1. RESULTS: Sixty-eight (73%) had small mutations, including 34 nonsense mutations, 27 microdeletions and insertions, and 7 splice site mutations. No missense mutations were found. One nonsense mutation in exon 59 was detected in four patients. Most mutations were new; 54 of 62 different small mutations have not been reported. Mutations were found throughout the gene: 24% in the first quartile, 31% in the second, 16% in the third, and 29% in the fourth. CONCLUSIONS: A highly sensitive single-strand conformation polymorphism method substantially increased detection of small dystrophin gene mutations and made it possible to diagnose approximately 90% of patients with Duchenne dystrophy by DNA analysis. These findings, combined with cost savings and safety issues, provide compelling reasons to consider DNA analysis as the initial diagnostic test for the suspected dystrophin-deficient patient.

Dystrophin expression in a Duchenne muscular dystrophy patient with a frame shift deletion.

The exon 45 deletion is a common dystrophin gene deletion. Although this is an out-of-frame deletion, which should not allow for protein synthesis, it has been observed in mildly affected patients. We describe a patient with an exon 45 deletion who produced protein, but still had a severe Duchenne muscular dystrophy phenotype. RT-PCR analysis and cDNA sequencing from the muscle biopsy sample revealed that the exon 45 deletion induced exon skipping of exon 44, which resulted in an in-frame deletion and the production of dystrophin. A conformational change in dystrophin induced by the deletion is proposed as being responsible for the severe phenotype in the patient. We feel that the variable clinical phenotype observed in patients with the exon 45 deletion is not due to exon splicing but may be the result of other environmental or genetic factors, or both.

Detection of cytomegalovirus in liver transplant biopsies. A comparison of light microscopy, immunohistochemistry, duplex PCR and nested PCR.

The polymerase chain reaction was used to detect cytomegalovirus (CMV) in 91 formalin-fixed paraffin-embedded needle biopsies from 38 liver transplant patients with allograft dysfunction. Thirty donor liver biopsies served as negative controls. PCR results were compared with light microscopy (LM), immunohistochemical staining (IH) for CMV early and late antigen, and clinical data. Primers to the major immediate early gene (MIE) and the viral DNA polymerase gene were duplex amplified. PCR product was reamplified with a nested primer set for the MIE and confirmed by electrophoretic mobilities and dot blotting. Primers for human beta-hemoglobin were used as internal controls. Seventeen of 38 patients had clinical evidence of cytomegalovirus disease, 12 of these were IH-positive, 14 were LM-positive, 15 were duplex PCR-positive and 17 were nested PCR-positive. In addition, duplex PCR was positive in one patient without other evidence of CMV disease, while nested PCR was positive in 12 such patients. The sensitivity and negative predictive value of nested PCR was 100%--however, the specificities and positive predictive values were only 42.9 and 58.6%, respectively. The control group was completely negative by LM, IH, and duplex PCR, however, 6 of 30 patients were nested PCR-positive. The number of nested-positive, duplex-negative patients without CMV disease was significantly greater in the transplant group versus the control group (12/21 vs. 6/30, P < 0.009). The incidence of IgG seropositivity was also significantly greater in the transplant group versus the controls (29/32 vs. 15/24, P < 0.02). We conclude that nested PCR may be an overly sensitive technique for the detection of clinically relevant CMV disease. A negative nested PCR assay for CMV may, however, help rule-out symptomatic CMV infection in an individual case. Duplex PCR showed little advantage over LM, while IH was confirmatory but did not add any new information in this study.

Identification of a missense mutation in a Friedreich's ataxia patient: implications for diagnosis and carrier studies.

Approximately 95% of all Friedreich's ataxia (FA) patients are homozygous for a large GAA triplet-repeat expansion in the first intron of the Friedreich's ataxia gene (FRDA). The remaining cases are expected to be compound heterozygous with a GAA expansion on one allele and a point mutation on the other. Generally, the clinical diagnostic profile in this group of patients is indistinguishable from that in classic FA patients with homozygous expansions. This study describes a mildly affected patient who presents with only one expanded allele by Southern blot analysis. Point mutation screening shows a single base change in FRDA exon 3 resulting in a nonconservative amino acid replacement in the N-terminal portion of the frataxin protein. Extended family studies show that two of the patient's sibs are carriers of the expanded allele and one is a carrier of the missense mutation. This case study demonstrates the benefits of implementing a combined Southern blot and point mutation diagnostic protocol for compound heterozygous patients. By identifying both mutations, this procedure confirms the diagnosis of FA in patients with an atypical disease course and allows for more complete family studies.

Spinal muscular atrophy variant with congenital fractures.

A single report of brothers born to first-cousin parents with a form of acute spinal muscular atrophy (SMA) and congenital fractures suggested that this combination represented a distinct form of autosomal recessive SMA. We describe a boy with hypotonia and congenital fractures whose sural nerve and muscle biopsies were consistent with a form of spinal muscular atrophy. Molecular studies identified no abnormality of the SMN(T) gene on chromosome 5. This case serves to validate the suggestion of a distinct and rare form of spinal muscular atrophy while not excluding possible X-linked inheritance.

Heteroduplex analysis of the dystrophin gene: application to point mutation and carrier detection.

Approximately one-third of the Duchenne muscular dystrophy patients have undefined mutations in the dystrophin gene. For carrier and prenatal studies in families without detectable mutations, the indirect restriction fragment length polymorphism linkage approach is used. Using a multiplex amplification and heteroduplex analysis of dystrophin exons, we identified nonsense mutations in two DMD patients. Although the nonsense mutations are predicted to severely truncate the dystrophin protein, both patients presented with mild clinical courses of the disease. As a result of identifying the mutation in the affected boys, direct carrier studies by heteroduplex analysis were extended to other relatives. We conclude that the technique is not only ideal for mutation detection but is also useful for diagnostic testing.

Germline mosaicism at the fragile X locus.

We have identified a fragile X syndrome pedigree where the disorder is associated with a molecular deletion. The deletion was present in the DNA of 2 sons but was absent in the mother's somatic cell (lymphocyte) DNA. The results are consistent with the deletion arising as a postzygotic event in the mother, who therefore is germinally mosaic. This finding has important implications for counseling fragile X families with deletion mutations.

Increased D allele frequency of the angiotensin-converting enzyme gene in pulmonary fibrosis.

An insertion/deletion (I/D) polymorphism of the angiotensin-converting enzyme has previously been studied extensively in relationship to cardiovascular and renal disease. The deletion/deletion (D/D) genotype is associated with a poor outcome in immunoglobulin (Ig) A nephropathy. However, the association of this genetic marker in cardiovascular and renal disease has generated controversy, with the exception of the rate of progression and therapeutic responsiveness in IgA nephropathy. Many of the same cytokines and polypeptide mediators involved in fibrosis of the cardiovascular and renal systems have been shown to be involved in pulmonary fibrosis. We examined the I/D polymorphism of the angiotensin-converting enzyme in a group of 24 patents with interstitial pneumonia and moderate to severe pulmonary fibrosis defined by radiographic studies, pulmonary function tests, and histologic findings. The incidence of the D allele in this study population was 69.0%, which is approximately 15.0% higher than the incidence in the general population of 54.0%. The incidence of the D/D genotype was 42.0%, which is approximately 11.0% greater than that in the general population (31.0%). The distribution of the D/D, I/D, and insertion/insertion genotypes of these 24 patients was not significantly different from that of historical controls (P =.1; chi(2) test); there were marginally significantly more D alleles among the 48 observed alleles than would be expected (P =.04).

Diffuse leiomyomatosis of the uterus: a case report with clonality analysis.

Diffuse uterine leiomyomatosis is a rare condition distinguished from the common uterine leiomyomata by involvement of the entire myometrium by innumerable, ill-defined, often small and confluent, histologically benign smooth-muscle nodules. Fourteen cases have been previously described in the literature. We report a case of diffuse leiomyomatosis in a 39-year-old woman. Several microscopic foci of the process were microdissected for clonality analysis. All samples showed a non-random X-chromosome inactivation pattern, and thus were consistent with a monoclonal neoplastic population of cells. However, in different foci of tumor, different X chromosomes were inactivated, supporting the independent origin of neoplastic clones and rejecting the possibility of a single clonal origin of all tumor cells. The results of the molecular analysis suggest that diffuse uterine leiomyomatosis may be an exuberant example of diffuse and uniform involvement of the entire myometrium by multiple leiomyomata. HUM PATHOL 31:1429-1432.

Prevalence of the factor VLeiden mutation among autopsy patients with pulmonary thromboembolic disease using an improved method for factor VLeiden detection.

Activated protein C resistance caused by factor VLeiden mutation is the most common inherited predisposing cause of venous thromboembolism, including pulmonary embolism (PE). We studied whether the incidence of factor VLeiden is higher among patients with PE evident at autopsy than in the general population. Paraffin-embedded fixed tissue blocks from all autopsy patients with diagnosed pulmonary thromboembolic disease during a 4-year period were collected for DNA extraction. Extraction and molecular analysis of the DNA was performed with an improved technique with an internal control to determine the presence of factor VLeiden mutation. Analysis of 82 autopsy cases with PE yielded 5 patients who were heterozygotes. Seventy-seven of the 82 patients analyzed were normal, and no homozygotes for factor VLeiden mutation were identified. This yielded a positive rate of 6% overall and 7% among white patients, which is similar to the incidence of heterozygotes in the white population. This study indicates that routine determination of factor VLeiden mutation is not warranted for patients with PE diagnosed at autopsy.

Optimization of DNA extraction from formalin-fixed tissue and its clinical application in Duchenne muscular dystrophy.

The authors report a case of Duchenne muscular dystrophy in which a deletion was determined by multiplex polymerase chain reaction using postmortem tissue from a proband who died in 1985, 2 years before the cloning of the dystrophin gene. Several extraction methods were analyzed to determine optimal conditions for recovery of DNA from fixed tissue. Variables examined were tissue lysis times and temperatures, a simple salting-out procedure for purification of DNA, polymerase chain reaction amplification of crude lysate versus purified DNA, and lysis of different tissues and tissue quantities. Extracted DNA was analyzed spectrophotometrically, electrophoretically, and by its suitability for polymerase chain reaction amplification using exon flanking primers of the dystrophin gene. Lysis at 37 degrees C for less than 24 hours led to the recovery of predominantly low-molecular-weight DNA, which was unsuitable for polymerase chain reaction in this experience. Lysis at 54 degrees C consistently yielded visible, spoolable quantities of high-molecular-weight DNA in as little as 24 hours. Direct amplification of crude, unpurified lysates was unsuccessful. However, purified samples yielded consistent amplification products. The purification step was simplified by substituting a rapid salting-out procedure for organic extractions.

Transfusion-associated graft-vs-host disease. A fatal case caused by blood from an unrelated HLA homozygous donor.

Transfusion-associated graft-vs-host disease (TA-GVHD) is a rare complication of transfusion. We report fatal TA-GVHD in a 63-year-old coronary artery bypass patient of European descent after an RBC transfusion from an unrelated donor. The patient had mild lymphocytopenia and received 2 80-mg doses of methylprednisolone and 7 units of RBCs. On day 14 after the transfusion, he had fever, elevated liver enzyme levels, and a macular rash. Pancytopenia and bone marrow aplasia developed. On day 26, he had a massive gastrointestinal hemorrhage and died. At autopsy, histopathologic findings of the skin, liver, bone marrow, and gastrointestinal tract were consistent with TA-GVHD. One donor of the transfused RBCs (3 days old at transfusion) had a 1-way HLA match with the patient. A method using multiplex polymerase chain reaction is presented. This patient with TA-GVHD and mild immune suppression suggests that blood component irradiation guidelines may need to be reevaluated.

The incidence of the gene for thermolabile methylene tetrahydrofolate reductase in African Americans.

Increased levels of homocysteine have been linked to both arterial and venous thromboembolic problems (1,2). Homocystinuria is a relatively rare disorder caused by a deficiency of cystathione synthase and is characterized by markedly increased levels of homocysteine and premature vascular disease (3-5). Epidemiological studies have suggested that mild elevations of homocysteine are also associated with vascular disease (2). Recent evidence suggests that a polymorphism of the gene encoding for 5,10-methylene tetrahydrofolate reductase (MTHFR) gives rise to a thermolabile form of the enzyme that is associated with increased levels of homocysteine when inherited as a homozygous trait (6). This polymorphism is due to a C --> T substitution at nucleotide 677 which converts an alanine to valine in a conserved portion of the molecule (6). The allele frequency for the thermolabile form of the enzyme was quite high (0.38) in a population of French Canadians. This polymorphism thus appears to be a common risk factor for increased plasma levels of homocysteine and vascular diseases. As the incidence of such genetic polymorphisms often varies among ethnic populations, we were interested in comparing the incidence of this polymorphism in Caucasians and African Americans.

A placebo-controlled trial of gabapentin in spinal muscular atrophy.

OBJECTIVE: To evaluate the efficacy of gabapentin in increasing muscle strength of patients with spinal muscular atrophy (SMA). BACKGROUND: Preclinical data in experimental models of motor neuron disease suggest a neuroprotective effect of gabapentin. METHODS: Gabapentin (1200 mg), or placebo, was administered three times daily in a randomized, double-blind trial for 12 months. The primary outcome measure was the average percent change from baseline, based on the measurement of strength in four muscles (elbow flexion and hand grip bilaterally) for each patient. Drug efficacy was examined by comparing the percent change in strength for patients on drug vs. placebo. Secondary efficacy variables included: forced vital capacity (FVC), SMA functional rating scale (SMAFRS), and mini-Sickness Impact Profile (SIP). RESULTS: Eighty-four patients, with type II or III SMA, were enrolled at eight sites across the United States. There were no differences in baseline features. There was no difference between the placebo and drug groups in any outcome measure. CONCLUSIONS: This study demonstrates the feasibility of this trial design and provides data for the design of future clinical trials in SMA.

Perspectives and molecular diagnosis of Duchenne and Becker muscular dystrophies.

Molecular genetic understanding of Duchenne and Becker muscular dystrophies has unfolded rapidly in the past decade. The new molecular understanding has enhanced diagnosis, prognosis, carrier detection, and prenatal diagnosis. Most importantly, strategies are being devised currently for the treatment of the disorder. This article provides an update on the molecular findings and their applicability in clinical practice.