Deficiency of the 50 kDa dystrophin-associated glycoprotein and abnormal expression of utrophin in two south Asian cousins with variable expression of severe childhood autosomal recessive muscular dystrophy.

Two male cousins with severe childhood, autosomal recessive, Duchenne-like, muscular dystrophy (SCARMD) have been identified with a deficiency of the 50 kDa dystrophin-associated glycoprotein but normal expression of dystrophin. Both boys were from consanguineous marriages and were Asian, having originated from Pakistan. This is in contrast to all previously reported cases from North Africa. Clinical severity varied and the patients were still able to walk at 13 and 12 yr, respectively. Neither had calf hypertrophy, a feature reported to be almost consistent in the North African patients. Abnormal expression of utrophin, the dystrophin-related protein, was observed on the surface of several non-regenerating muscle fibres, with less intense immunolabelling in the clinically more affected child. This family shows that SCARMD is not confined to North Africa and illustrates a hitherto unreported expression of utrophin in this condition.

Two

    male
    GENDER

    cousins with severe childhood, autosomal recessive, Duchenne-like, muscular dystrophy (SCARMD)
    FAMILY

 have been identified with a

    deficiency of the 50 kDa dystrophin-associated glycoprotein
    BIOCHEMICAL

 but

    normal expression of dystrophin
    NEG_FINDINGS

. Both

    boys
    GENDER

    were from consanguineous marriages
    FAMILY

 and were

    Asian
    ETHNICITY

, having originated from

    Pakistan
    ETHNICITY

. This is in contrast to all previously reported cases from

    North Africa
    ETHNICITY

. Clinical severity varied and the patients were

    still able to walk at 13 and 12 yr
    SYMPTOMS

, respectively. Neither had

    calf hypertrophy
    SYMPTOMS

, a feature reported to be almost consistent in the

    North African
    ETHNICITY

 patients.

    Abnormal expression of utrophin, the dystrophin-related protein, was observed on the surface of several non-regenerating muscle fibres
    MICROSCOPIC

, with

    less intense immunolabelling
    SYMPTOMS

 in the clinically more affected child. This family shows that

    SCARMD
    DIAGNOSIS

 is not confined to

    North Africa
    ETHNICITY

 and illustrates a hitherto unreported expression of

    utrophin
    BIOCHEMICAL

 in this condition.

Asian
Pakistan
North Africa
North African
North Africa

still able to walk at 13 and 12 yr
calf hypertrophy
less intense immunolabelling


Complex genetic counseling and exclusion of Duchenne muscular dystrophy in a twin pregnancy after in vitro fertilization (IVF).

A twin pregnancy following in vitro fertilization-embryo transfer coincidentally at risk for the X-linked recessive Duchenne muscular dystrophy is described. First-trimester prenatal diagnosis by transabdominal chorionic villus samplings on the dichorionic placentae and molecular linkage analysis could exclude the disorder in both fetuses. Genetic counseling and prenatal diagnosis were particularly complex due to the twin pregnancy, the need for linkage analysis, and confined placental mosaicism 45,X/46XX in one of the fetuses. All parties should be aware that additional invasive diagnostic procedures in the second trimester might be required. It is proposed that, in similar situations, only one, arguably two, fertilized egg be transferred at a time to facilitate prenatal diagnosis and decision making for these rare couples. This problem, however, may be increasingly overcome by preimplantation diagnosis.

A

    twin pregnancy
    DIAGNOSIS

 following

    in vitro fertilization-embryo transfer
    MEDICATION

 coincidentally at risk for the

    X-linked recessive Duchenne muscular dystrophy
    GENETICS

 is described.

    First-trimester prenatal diagnosis
    PARACLINICAL

 by

    transabdominal chorionic villus samplings on the dichorionic placentae
    PARACLINICAL

 and

    molecular linkage analysis
    PARACLINICAL

 could exclude

    the disorder in both fetuses
    FAMILY

.

    Genetic counseling
    MEDICATION

 and

    prenatal diagnosis
    PARACLINICAL

 were particularly complex due to the

    twin pregnancy
    DIAGNOSIS

, the need for

    linkage analysis
    PARACLINICAL

, and

    confined placental mosaicism 45,X/46XX in one of the fetuses
    GENETICS

. All parties should be aware that additional invasive diagnostic procedures in the second trimester might be required. It is proposed that, in similar situations, only one

    , arguably two, fertilized egg be transferred
    MEDICATION

 at a time to facilitate prenatal diagnosis and decision making for these rare couples. This problem, however, may be increasingly overcome by

    preimplantation diagnosis
    MEDICATION

.


Restoration of half the normal dystrophin sequence in a double-deletion Duchenne muscular dystrophy family.

Two male cousins with Duchenne muscular dystrophy were found to have different maternal dystrophin gene haplotypes and different deletion mutations. One propositus showed two noncontiguous deletions--one in the 5', proximal deletional hotspot region, and the other in the 3', more distal deletional hotspot region. The second propositus showed only the 5' deletion. Using multiple fluorescent exon dosage and fluorescent multiplex CA repeat linkage analyses, we show that the mother of each propositus carries both deletions on the same grandmaternal X chromosome. This paradox is explained by a single recombinational event between the 2 deleted regions of one of the carrier's dystrophin genes, giving rise to a son with a partially "repaired" gene retaining only the 5' deletion.

Two

    male
    GENDER

    cousins with Duchenne muscular dystrophy
    FAMILY

 were found to have

    different maternal dystrophin gene haplotypes
    GENETICS

 and

    different deletion mutations
    GENETICS

. One propositus showed

    two noncontiguous deletions--one in the 5', proximal deletional hotspot region, and the other in the 3', more distal deletional hotspot region
    GENETICS

. The second propositus showed

    only the 5' deletion
    GENETICS

. Using

    multiple fluorescent exon dosage and fluorescent multiplex CA repeat linkage analyses
    PARACLINICAL

, we show that

    the mother of each propositus carries both deletions on the same grandmaternal X chromosome
    FAMILY

. This paradox is explained by a

    single recombinational event between the 2 deleted regions of one of the carrier's dystrophin genes
    GENETICS

, giving rise to a

    son with a partially "repaired" gene retaining only the 5' deletion
    FAMILY

.


A novel point mutation (G-1 to T) in a 5' splice donor site of intron 13 of the dystrophin gene results in exon skipping and is responsible for Becker muscular dystrophy.

The mutations in one-third of Duchenne and Becker muscular dystrophy patients remain unknown, as they do not involve gross rearrangements of the dystrophin gene. We now report a defect in the splicing of precursor mRNA (pre-mRNA), resulting from a maternally inherited mutation of the dystrophin gene in a patient with Becker muscular dystrophy. This defect results from a G-to-T transversion at the terminal nucleotide of exon 13, within the 5' splice site of intron 13, and causes complete skipping of exon 13 during processing of dystrophin pre-mRNA. The predicted polypeptide encoded by the aberrant mRNA is a truncated dystrophin lacking 40 amino acids from the amino-proximal end of the rod domain. This is the first report of an intraexon point mutation that completely inactivates a 5' splice donor site in dystrophin pre-mRNA. Analysis of the genomic context of the G-1-to-T mutation at the 5' splice site supports the exon-definition model of pre-mRNA splicing and contributes to the understanding of splice-site selection.

The mutations in one-third of

    Duchenne
    DIAGNOSIS

 and

    Becker muscular dystrophy
    DIAGNOSIS

 patients remain unknown, as they do not involve

    gross rearrangements of the dystrophin gene
    NEG_FINDINGS

. We now report a

    defect in the splicing of precursor mRNA (pre-mRNA)
    GENETICS

, resulting from a

    maternally inherited mutation of the dystrophin gene
    GENETICS

 in a patient with

    Becker muscular dystrophy
    DIAGNOSIS

. This defect results from

    a G-to-T transversion at the terminal nucleotide of exon 13, within the 5' splice site of intron 13
    GENETICS

, and causes

    complete skipping of exon 13 during processing of dystrophin pre-mRNA
    GENETICS

. The predicted polypeptide encoded by the

    aberrant mRNA
    GENETICS

 is a

    truncated dystrophin lacking 40 amino acids from the amino-proximal end of the rod domain
    GENETICS

. This is the first report of an

    intraexon point mutation that completely inactivates a 5' splice donor site in dystrophin pre-mRNA
    GENETICS

. Analysis of the genomic context of the

    G-1-to-T mutation at the 5' splice
    GENETICS

 site supports the exon-definition model of pre-mRNA splicing and contributes to the understanding of splice-site selection.

Duchenne
Becker muscular dystrophy
Becker muscular dystrophy


Identification of a point mutation and germinal mosaicism in a Duchenne muscular dystrophy family.

Duchenne and Becker muscular dystrophies (DMD and BMD) are allelic X-linked disorders arising from mutations in the (2.4 Mb) dystrophin gene at Xp21. We have applied the reverse transcriptase-polymerase chain reaction (RT-PCR) to identify a larger than normal dystrophin mRNA from a male with Duchenne muscular dystrophy and his younger affected brother. The increased size of the dystrophin mRNA was due to a splice-site mutation at the exon 26:intron 26 junction where a T to G substitution prevented normal RNA processing. A cryptic splice-site, downstream of the mutation, was activated during processing, resulting in the inclusion of 117 bases of intron 26. This insertion introduced an in-frame stop codon into the mature dystrophin mRNA. An allele-specific test was developed to identify the mutation and was applied to this family. Interestingly, the mother of the two affected boys did not carry the mutation, as determined by allele-specific amplification and direct DNA sequence analysis, indicating gonadal mosaicism. Her eldest daughter, designated as a carrier based upon conventional testing and haplotype analysis, also did not carry the family mutation. Initial haplotyping of the family appeared to be straightforward with gonadal mosaicism becoming evident only after allele-specific analysis. The application of linked markers to identify the disease allele for conventional genetic counselling would have been erroneous in this family and highlights the diagnostic power of precise identification of the disease-causing mutation.

    Duchenne and Becker muscular dystrophies (DMD and BMD)
    DIAGNOSIS

 are

    allelic X-linked disorders
    GENETICS

 arising from

    mutations in the (2.4 Mb) dystrophin gene at Xp21
    GENETICS

. We have applied the

    reverse transcriptase-polymerase chain reaction (RT-PCR)
    PARACLINICAL

 to identify a

    larger than normal dystrophin mRNA
    MICROSCOPIC

 from a

    male
    GENDER

 with

    Duchenne muscular dystrophy
    DIAGNOSIS

 and

    his
    GENDER

 younger affected

    brother
    GENDER

. The

    increased size of the dystrophin mRNA
    MICROSCOPIC

 was due to a

    splice-site mutation at the exon 26:intron 26 junction where a T to G substitution prevented normal RNA processing. A cryptic splice-site, downstream of the mutation, was activated during processing, resulting in the inclusion of 117 bases of intron 26
    GENETICS

. This

    insertion introduced an in-frame stop codon into the mature dystrophin mRNA
    GENETICS

. An

    allele-specific test
    PARACLINICAL

 was developed to identify the mutation and was applied to this family. Interestingly,

    the mother of the two affected boys did not carry the mutation
    FAMILY

, as determined by

    allele-specific amplification and direct DNA sequence analysis
    PARACLINICAL

, indicating

    gonadal mosaicism
    FAMILY

.

    Her eldest daughter, designated as a carrier
    FAMILY

 based upon

    conventional testing and haplotype analysis
    FAMILY

,

    also did not carry the family mutation
    FAMILY

. Initial

    haplotyping of the family
    PARACLINICAL

 appeared to be straightforward with

    gonadal mosaicism
    SYMPTOMS

 becoming evident only after

    allele-specific analysis
    PARACLINICAL

. The application of linked markers to identify the disease allele for

    conventional genetic counselling
    MEDICATION

 would have been erroneous in this family and highlights the diagnostic power of precise identification of the disease-causing mutation.


Molecular diagnosis of Duchenne muscular dystrophy by use of a conformational polymorphism in the absence of DNA from an affected boy.

We report the molecular diagnosis of Duchenne muscular dystrophy (DMD) in an extended pedigree by use of a conformational polymorphism detected by the SSCP method, which allowed direct prenatal diagnosis and carrier detection while no DNA from an affected boy was available.

We report the

    molecular diagnosis
    PARACLINICAL

 of

    Duchenne muscular dystrophy (DMD)
    DIAGNOSIS

 in an extended pedigree by use of a

    conformational polymorphism detected by the SSCP method
    PARACLINICAL

, which allowed

    direct prenatal diagnosis
    PARACLINICAL

 and

    carrier detection
    PARACLINICAL

 while

    no DNA from an affected boy was available
    NEG_FINDINGS

.


Isolated glycerol kinase deficiency in a neonate.

Glycerol kinase deficiency occurs either as a relatively benign isolated enzyme deficiency, or as part of a syndrome resulting from a microdeletion in the p21 region of the X chromosome associated with congenital adrenal hypoplasia and/or Duchenne muscular dystrophy. Developmental delay is a consistent feature of the microdeletion syndrome but not of the isolated enzyme defect. We report a case of isolated glycerol kinase deficiency in a neonate presenting with hypotonia, apnea, mild developmental delay, and glyceroluria, without evidence of adrenal insufficiency or myopathy. A mild communicating hydrocephalus was noted on magnetic resonance imaging brain scan. It is important, therefore, to exclude glyceroluria in infants being investigated for apnea and hypotonia.

    Glycerol kinase deficiency
    BIOCHEMICAL

 occurs either as a relatively benign

    isolated enzyme deficiency
    BIOCHEMICAL

, or as part of a syndrome resulting from a

    microdeletion in the p21 region of the X chromosome
    GENETICS

 associated with

    congenital adrenal hypoplasia
    DIAGNOSIS

 and/or

    Duchenne muscular dystrophy
    DIAGNOSIS

.

    Developmental delay
    SYMPTOMS

 is a consistent feature of the

    microdeletion syndrome
    DIAGNOSIS

 but not of the

    isolated enzyme defect
    BIOCHEMICAL

. We report a case of

    isolated glycerol kinase deficiency
    BIOCHEMICAL

 in a

    neonate
    AGE

 presenting with

    hypotonia
    SYMPTOMS

,

    apnea
    SYMPTOMS

,

    mild developmental delay
    SYMPTOMS

, and

    glyceroluria
    BIOCHEMICAL

,

    without evidence of adrenal insufficiency or myopathy
    NEG_FINDINGS

. A

    mild communicating hydrocephalus
    SYMPTOMS

 was noted on

    magnetic resonance imaging brain scan
    PARACLINICAL

. It is important, therefore, to exclude

    glyceroluria
    NEG_FINDINGS

 in

    infants
    AGE

 being investigated for

    apnea
    SYMPTOMS

 and

    hypotonia
    SYMPTOMS

.

Glycerol kinase deficiency
isolated enzyme deficiency
isolated enzyme defect
isolated glycerol kinase deficiency
glyceroluria

congenital adrenal hypoplasia
Duchenne muscular dystrophy
microdeletion syndrome

Developmental delay
hypotonia
apnea
mild developmental delay
mild communicating hydrocephalus
apnea
hypotonia


X-linked lymphoproliferative disease. Detection of a paternally inherited mutation in a German family using haplotype analysis.

To study the inheritance of the X-linked lymphoproliferative disease (XLP) locus in a German family.

Haplotype segregation analysis.

Departments of Pediatrics and Human Genetics, University of Würzburg and University of Ulm, Federal Republic of Germany.

Fourteen members of a family with XLP.

None.

Using molecular genetic techniques, we diagnosed the XLP status of the members of a German family. Restriction fragment length polymorphism analysis with closely linked polymorphic X-chromosomal DNA markers (Xq25-q27) revealed XLP carrier status in a female infant. Moreover, the XLP mutation was suggested to have occurred in the germ cell of the grandfather.

This is the first report of a paternally inherited XLP mutation. The recurrence risk for germ cell mosaicism in XLP may be similar to that of X-linked Duchenne muscular dystrophy.

To study the

    inheritance of the X-linked lymphoproliferative disease (XLP) locus
    GENETICS

 in a

    German
    ETHNICITY

 family.

    Haplotype segregation analysis
    PARACLINICAL

.

    Departments of Pediatrics
    PARACLINICAL

 and

    Human Genetics
    PARACLINICAL

, University of

    Würzburg
    ETHNICITY

 and University of Ulm,

    Federal Republic of Germany
    ETHNICITY

.

    Fourteen members of a family with XLP
    FAMILY

.

Using

    molecular genetic techniques
    PARACLINICAL

, we diagnosed the

    XLP
    DIAGNOSIS

 status of the members of a

    German
    ETHNICITY

 family.

    Restriction fragment length polymorphism analysis with closely linked polymorphic X-chromosomal DNA markers (Xq25-q27)
    PARACLINICAL

 revealed

    XLP carrier
    DIAGNOSIS

 status in a

    female
    GENDER

    infant
    AGE

. Moreover,

    the XLP mutation was suggested to have occurred in the germ cell of the grandfather
    GENETICS

.

This is the first report of a

    paternally inherited XLP mutation
    GENETICS

. The recurrence risk for

    germ cell mosaicism
    GENETICS

 in

    XLP
    DIAGNOSIS

 may be similar to that of

    X-linked
    GENETICS

    Duchenne muscular dystrophy
    DIAGNOSIS

.

German
Würzburg
Federal Republic of Germany
German

XLP
XLP carrier
XLP
Duchenne muscular dystrophy


Duchenne muscular dystrophy associated with fetal pleural effusion and polyhydramnios.

A case of fetal pleural effusion in a fetus affected with Duchenne muscular dystrophy (DMD) is reported. This case is discussed in the context of the previous observation of frequent stillbirths among male fetuses in DMD families.

A case of

    fetal pleural effusion
    SYMPTOMS

 in a

    fetus
    AGE

 affected with

    Duchenne muscular dystrophy (DMD)
    DIAGNOSIS

 is reported. This case is discussed in the context of the previous observation of frequent

    stillbirths
    SYMPTOMS

 among

    male
    GENDER

 fetuses in

    DMD
    DIAGNOSIS

 families.


Isolation of a yeast artificial chromosome contig spanning the X chromosomal translocation breakpoint in a patient with Rett syndrome.

Rett syndrome is a neurodevelopmental disorder observed exclusively in females. A de novo X;3 translocation was detected in a patient (TH) with Rett syndrome. The X chromosomal breakpoint maps to Xp21.3 between the distal end of the Duchenne muscular dystrophy (DMD) gene and the DXS28 (C7) locus. To determine if this translocation caused the Rett syndrome in this patient, our efforts focused on mapping and cloning of the X chromosomal breakpoint in this patient. Toward these goals, we generated a set of radiation-reduced hybrid cell lines for the short arm of the X chromosome to use as a source for region-specific markers. Using Alu-PCR, 13 new DNA markers were isolated from a radiation-reduced hybrid, which retained both DMD and DXS28. These markers were localized within Xp21 using DNA from males with various interstitial deletions in this region. Two new markers, K23-2p and K23b-1, were found to be closer flanking markers to the X chromosomal breakpoint than DMD and DXS28. Long range restriction mapping using K23-2p and K23b-1 determined that the maximum distance between them was 800 kb. Several of the new markers were developed into sequence tagged-sites and were used to isolate yeast artificial chromosome (YAC) clones. A total of 22 YAC clones was isolated and characterized; these YACs were then developed into 3 large contigs in the Xp21.3 region. This effort resulted in the cloning of the region containing the X chromosomal translocation breakpoint of the Rett syndrome patient in a 170-kb YAC clone.

    Rett syndrome
    DIAGNOSIS

 is a

    neurodevelopmental disorder
    DIAGNOSIS

 observed exclusively in

    females
    GENDER

. A

    de novo X;3 translocation
    GENETICS

 was detected in a patient (

    TH
    GENDER

) with

    Rett syndrome
    DIAGNOSIS

. The

    X chromosomal breakpoint maps to Xp21.3 between the distal end of the Duchenne muscular dystrophy (DMD) gene and the DXS28 (C7) locus
    GENETICS

. To determine if this translocation caused the

    Rett syndrome
    DIAGNOSIS

 in this patient, our efforts focused on mapping and cloning of the X chromosomal breakpoint in this patient. Toward these goals, we generated a set of

    radiation-reduced hybrid cell lines for the short arm of the X chromosome
    MICROSCOPIC

 to use as a source for region-specific markers. Using

    Alu-PCR
    PARACLINICAL

, 13 new DNA markers were isolated from a

    radiation-reduced hybrid
    MICROSCOPIC

, which

    retained both DMD and DXS28
    GENETICS

. These markers were localized within

    Xp21
    GENETICS

 using DNA from

    males
    GENDER

 with various

    interstitial deletions in this
    GENETICS

 region. Two new markers,

    K23-2p and K23b-1
    GENETICS

, were found to be closer flanking markers to the

    X chromosomal breakpoint than DMD and DXS28
    GENETICS

.

    Long range restriction mapping
    PARACLINICAL

 using K23-2p and K23b-1 determined that the maximum distance between them was 800 kb. Several of the new markers were developed into sequence tagged-sites and were used to isolate yeast artificial chromosome (YAC) clones. A total of 22 YAC clones was isolated and characterized; these YACs were then developed into 3 large contigs in the Xp21.3 region. This effort resulted in the cloning of the region

    containing the X chromosomal translocation breakpoint
    GENETICS

 of the

    Rett syndrome
    DIAGNOSIS

 patient in a 170-kb YAC clone.

Rett syndrome
neurodevelopmental disorder
Rett syndrome
Rett syndrome
Rett syndrome


Molecular analysis of the Duchenne muscular dystrophy gene in patients with Becker muscular dystrophy presenting with dilated cardiomyopathy.

Molecular analysis of the Duchenne muscular dystrophy (DMD) gene was performed on 4 unrelated patients with Becker muscular dystrophy (BMD) presenting with dilated cardiomyopathy. Two patients with a deletion involving exon 1 were quite unique in that they developed fatal myocardial involvement in their teens, despite the absence of significant muscular weakness. The deletion found in these patients comprised the 3'-end of exon 1 and the greater part of intron 1. Two other patients with a deletion of exon 47 showed progressive muscular atrophy and weakness; they were considered to be typical BMD in both clinical features and the type of gene deletion. We speculate that a deletion around exon 1 may severely damage the expression and/or the function of dystrophin selectively in cardiac muscle, but not in skeletal muscle.

    Molecular analysis of the Duchenne muscular dystrophy (DMD) gene
    PARACLINICAL

 was performed on 4 unrelated patients with

    Becker muscular dystrophy (BMD)
    DIAGNOSIS

 presenting with

    dilated cardiomyopathy
    DIAGNOSIS

. Two patients with a

    deletion involving exon 1
    GENETICS

 were quite unique in that they developed

    fatal myocardial involvement
    SYMPTOMS

    in their teens
    AGE

,

    despite
    NEG_FINDINGS

 the

    absence of significant muscular weakness
    NEG_FINDINGS

. The

    deletion found
    GENETICS

 in these patients

    comprised the 3'-end of exon 1 and the greater part of intron 1
    GENETICS

. Two other patients with a

    deletion of exon 47
    GENETICS

 showed

    progressive muscular atrophy
    SYMPTOMS

 and

    weakness
    SYMPTOMS

; they were considered to be typical

    BMD
    DIAGNOSIS

 in both clinical features and the type of gene deletion. We speculate that a

    deletion around exon 1
    GENETICS

 may severely damage the expression and/or the function of dystrophin selectively in

    cardiac muscle
    SYMPTOMS

, but

    not in skeletal muscle
    NEG_FINDINGS

.

Becker muscular dystrophy (BMD)
dilated cardiomyopathy
BMD

fatal myocardial involvement
progressive muscular atrophy
weakness
cardiac muscle


Quantitative analysis of dystrophin gene amplification products using a PC-based image analysis system.

A PC-based image analysis system for gel photographs of DNA gel patterns was developed. It was originally designed as a general-purpose, low-cost, yet high-performance system for wide applications in the biomedical area. In this study, we performed analysis of gel images obtained by polymerase chain reaction (PCR) amplification. The system employs a high-resolution CCD camera that can accurately measure grayness of the gel photographs for quantitative analysis of PCR products. The target DNA (exon 52 of the dystrophin gene), which had been found to be deleted in some patients with Duchenne/Becker muscular dystrophy (DMD/BMD), was amplified for the female family members together with the control DNA (exon 60) as a reference. The ratio of the target DNA to the control DNA was determined from PCR products to identify the carrier status of this disease by means of gene dosage. We conclude that 'the PC-based image analysis system' was useful for quantitative deletion analysis of DMD/BMD heterozygotes.

A

    PC-based image analysis system
    PARACLINICAL

 for

    gel photographs of DNA gel patterns
    PARACLINICAL

 was developed. It was originally designed as a general-purpose, low-cost, yet high-performance system for wide applications in the biomedical area. In this study, we performed

    analysis of gel images
    PARACLINICAL

 obtained by

    polymerase chain reaction (PCR) amplification
    PARACLINICAL

. The system employs a

    high-resolution CCD camera
    PARACLINICAL

 that can accurately measure

    grayness of the gel photographs
    PARACLINICAL

 for

    quantitative analysis of PCR products
    PARACLINICAL

. The target

    DNA (exon 52 of the dystrophin gene)
    GENETICS

, which had been found

    to be deleted
    GENETICS

 in some patients with

    Duchenne/Becker muscular dystrophy (DMD/BMD)
    DIAGNOSIS

, was

    amplified for the female family members
    PARACLINICAL

 together with the

    control DNA (exon 60)
    PARACLINICAL

 as a reference. The ratio of the target DNA to the control DNA was determined from

    PCR products
    PARACLINICAL

 to identify the carrier status of this disease by means of

    gene dosage
    PARACLINICAL

. We conclude that 'the

    PC-based image analysis system
    PARACLINICAL

' was useful for

    quantitative deletion analysis
    PARACLINICAL

 of

    DMD/BMD
    DIAGNOSIS

    heterozygotes
    GENETICS

.

DNA (exon 52 of the dystrophin gene)
to be deleted
heterozygotes


Negative-configuration electroretinogram in Oregon eye disease. Consistent phenotype in Xp21 deletion syndrome.

To determine whether abnormal configurations on electroretinogram were a consistent finding in patients with Xp21 deletion and to characterize the associated ophthalmologic phenotype.

Case series.

University hospitals and eye institutes.

Five patients with complex glycerol kinase deficiency (Duchenne-type or Becker's muscular dystrophy, glycerol kinase deficiency, and congenital adrenal hypoplasia) and demonstrated chromosomal deletions at Xp21. Control patients were matched by age.

Clinical information was obtained from medical records. Complete ophthalmologic examinations were performed. Electroretinography was performed using a Ganzfeld technique and chloral hydrate sedation.

We report the clinical features and abnormal configurations on electroretinograms of five patients with complex glycerol kinase deficiency, including follow-up studies on a previously described patient. The original patient had ocular hypopigmentation; four, strabismus; two, myopia; three, astigmatism; and one, symptomatic night blindness. All had negative configurations on scotopic electroretinograms showing a reduced-amplitude B wave in the dark-adapted state.

Our original report suggested a diagnosis of Aland Island eye disease, which appears to be an incomplete form of congenital stationary night blindness. Linkage data place Aland Island eye disease and congenital stationary night blindness at Xp11, whereas our patients had deletions at Xp21. The phenotype reported here may represent the effects of a single gene defect or the compound effects of the Xp21 contiguous gene syndrome (complex glycerol kinase deficiency). The phenotype is referred to as Oregon eye disease.

To determine whether

    abnormal configurations on electroretinogram
    SYMPTOMS

 were a consistent finding in patients with

    Xp21 deletion
    GENETICS

 and to characterize the associated ophthalmologic phenotype.

    University hospitals and eye institutes
    MEDICATION

.

Five patients with

    complex glycerol kinase deficiency
    BIOCHEMICAL

    (Duchenne-type or Becker's muscular dystrophy
    DIAGNOSIS

,

    glycerol kinase deficiency
    BIOCHEMICAL

, and

    congenital adrenal hypoplasia
    SYMPTOMS

) and demonstrated

    chromosomal deletions at Xp21
    GENETICS

. Control patients were matched by age.

Clinical information was obtained from

    medical records
    PARACLINICAL

.

    Complete ophthalmologic examinations
    PARACLINICAL

 were performed.

    Electroretinography
    PARACLINICAL

 was performed using a

    Ganzfeld technique
    PARACLINICAL

 and

    chloral hydrate sedation
    MEDICATION

.

We report the clinical features and

    abnormal configurations on electroretinograms
    SYMPTOMS

 of five patients with

    complex glycerol kinase deficiency
    BIOCHEMICAL

, including follow-up studies on a previously described patient. The original patient had

    ocular hypopigmentation
    SYMPTOMS

; four,

    strabismus
    SYMPTOMS

; two,

    myopia
    SYMPTOMS

; three,

    astigmatism
    SYMPTOMS

; and one,

    symptomatic night blindness
    SYMPTOMS

. All had

    negative configurations on scotopic electroretinograms showing a reduced-amplitude B wave in the dark-adapted state
    SYMPTOMS

.

Our original report suggested a diagnosis of

    Aland Island eye disease
    DIAGNOSIS

, which appears to be an incomplete form of

    congenital stationary night blindness
    SYMPTOMS

.

    Linkage data
    PARACLINICAL

 place

    Aland Island eye disease
    DIAGNOSIS

 and

    congenital stationary night blindness
    DIAGNOSIS

 at

    Xp11
    GENETICS

, whereas our patients had

    deletions at Xp21
    GENETICS

. The phenotype reported here may represent the effects of a

    single gene defect
    GENETICS

 or the

    compound effects of the Xp21 contiguous gene syndrome (complex glycerol kinase deficiency)
    GENETICS

. The phenotype is referred to as

    Oregon eye disease
    DIAGNOSIS

.

University hospitals and eye institutes
chloral hydrate sedation

complex glycerol kinase deficiency
glycerol kinase deficiency
complex glycerol kinase deficiency


[Quadriceps myopathy as dystrophin-associated myopathy].

Duchenne and Becker muscular dystrophy are both caused by a deletion in the dystrophin gene. In the past few years, many reports of atypical myopathies have been published where an association with a deletion within the same gene was found. We report one sporadic and one familial case of myopathy where we were able to demonstrate a deletion of the dystrophin locus using DNA analysis.

    Duchenne
    DIAGNOSIS

 and

    Becker muscular dystrophy
    DIAGNOSIS

 are both caused by a

    deletion in the dystrophin gene
    GENETICS

. In the past few years, many reports of

    atypical myopathies
    DIAGNOSIS

 have been published where an association with a

    deletion within the same gene
    GENETICS

 was found. We report one sporadic and one familial case of

    myopathy
    DIAGNOSIS

 where we were able to demonstrate a

    deletion of the dystrophin locus
    GENETICS

 using

    DNA analysis
    PARACLINICAL

.

Duchenne
Becker muscular dystrophy
atypical myopathies
myopathy

deletion in the dystrophin gene
deletion within the same gene
deletion of the dystrophin locus


Acute pericardial tamponade complicating spinal surgery in a child with Duchenne muscular dystrophy.

A hitherto unrecognised problem of pericardial tamponade complicating spinal surgery in a child with Duchenne muscular dystrophy is reported in this paper.

A hitherto unrecognised problem of

    pericardial tamponade complicating spinal surgery
    SYMPTOMS

 in a

    child
    AGE

 with

    Duchenne muscular dystrophy
    DIAGNOSIS

 is reported in this paper.


Occurrence of Duchenne dystrophy in Klinefelter's syndrome.

A boy with Duchenne muscular dystrophy and facial dysmorphism in conjunction with Klinefelter's genotype 47XXY is presented; this is an unusual situation with two genetic errors evolving over two generations. Karyotyping should be considered in boys with Duchenne muscular dystrophy who have unusual features.

A

    boy
    GENDER

 with

    Duchenne muscular dystrophy
    DIAGNOSIS

 and

    facial dysmorphism
    SYMPTOMS

 in conjunction with

    Klinefelter's genotype 47XXY
    GENETICS

 is presented; this is an unusual situation with

    two genetic errors evolving over two generations
    FAMILY

.

    Karyotyping
    PARACLINICAL

 should be considered in

    boys
    GENDER

 with

    Duchenne muscular dystrophy
    DIAGNOSIS

 who have

    unusual features
    SYMPTOMS

.

Duchenne muscular dystrophy
Duchenne muscular dystrophy


[Muscular dystrophy in a cat].

A case of muscular dystrophy in a 1-year-old male castrated Domestic Shorthair cat is presented. The most striking clinical features were regurgitation, a stiff gait, an increased muscle tone and exercise intolerance. Serum biochemistry panels showed a marked increase in the muscle specific enzyme creatine kinase, and moderately elevated levels of LDH, AST and ALT. Spontaneous electrical activity of skeletal muscles in the form of "bizarre high frequency discharges" and "myotonia-like repetitive discharges" were registered. Gross pathology revealed a marked hypertrophy of the skeletal muscles. The main histopathological changes were myofiber necrosis and calcification, variation in fiber size, hypertrophied muscle fibers of type I and type II and fiber splitting. Indirect immunofluorescence showed dystrophin deficiency. Feline muscular dystrophy resembles the X-linked human Duchenne muscular dystrophy (DMD). Besides the X-linked muscular dystrophy in the mouse and Golden Retriever the feline muscular dystrophy could represent another valuable animal model for the study of DMD.

A case of

    muscular dystrophy
    DIAGNOSIS

 in a

    1-year-old
    AGE

    male
    GENDER

    castrated Domestic Shorthair cat
    MEDICATION

 is presented. The most striking clinical features were

    regurgitation
    SYMPTOMS

, a

    stiff gait
    SYMPTOMS

, an

    increased muscle tone
    SYMPTOMS

 and

    exercise intolerance
    SYMPTOMS

.

    Serum biochemistry panels
    PARACLINICAL

 showed a

    marked increase in the muscle specific enzyme creatine kinase
    BIOCHEMICAL

, and

    moderately elevated levels of LDH, AST and ALT
    BIOCHEMICAL

.

    Spontaneous electrical activity of skeletal muscles in the form of "bizarre high frequency discharges" and "myotonia-like repetitive discharges"
    SYMPTOMS

 were registered.

    Gross pathology
    PARACLINICAL

 revealed a

    marked hypertrophy of the skeletal muscles
    SYMPTOMS

. The main histopathological changes were

    myofiber necrosis
    MICROSCOPIC

    and calcification
    SYMPTOMS

,

    variation in fiber size
    SYMPTOMS

,

    hypertrophied muscle fibers of type I and type II
    MICROSCOPIC

 and

    fiber splitting
    SYMPTOMS

.

    Indirect immunofluorescence
    PARACLINICAL

 showed

    dystrophin deficiency
    BIOCHEMICAL

.

    Feline muscular dystrophy
    DIAGNOSIS

 resembles the

    X-linked
    GENETICS

    human Duchenne muscular dystrophy (DMD)
    DIAGNOSIS

. Besides the

    X-linked
    GENETICS

    muscular dystrophy
    DIAGNOSIS

 in the mouse and

    Golden Retriever
    ETHNICITY

 the

    feline muscular dystrophy
    DIAGNOSIS

 could represent another valuable animal model for the study of

    DMD
    DIAGNOSIS

.

muscular dystrophy
Feline muscular dystrophy
human Duchenne muscular dystrophy (DMD)
muscular dystrophy
feline muscular dystrophy
DMD


Intrafamilial variability in dystrophin abundance correlated with difference in the severity of the phenotype.

In Duchenne muscular dystrophy, the progression of the disease is always severe and predictable, while in Becker dystrophy there is a wide variability (intra and inter familial) in the severity of the phenotype. We report here a family in which the proband, who is currently 15 years old, is showing a severe DMD progression, while his affected maternal uncle, aged 29, has a more benign course, compatible with BMD. No DNA deletion was detected in both patients. Dystrophin analysis through immunofluorescence and western blotting showed a negative pattern in the youngest patient and a positive one in the oldest. Apparently, this is the first report on intrafamilial variability in dystrophin abundance correlated with a difference in the severity of the phenotype.

In

    Duchenne muscular dystrophy
    DIAGNOSIS

, the progression of the disease is always severe and predictable, while in

    Becker dystrophy
    DIAGNOSIS

 there is a wide variability (intra and inter familial) in the severity of the phenotype. We report here a family in which the proband, who is

    currently 15 years old
    AGE

, is showing a

    severe DMD progression
    SYMPTOMS

, while

    his affected maternal uncle, aged 29, has a more benign course, compatible with BMD
    FAMILY

.

    No DNA deletion was detected
    NEG_FINDINGS

 in both patients.

    Dystrophin analysis through immunofluorescence and western blotting
    PARACLINICAL

 showed a negative pattern in the youngest patient and a positive one in the oldest. Apparently, this is the first report on

    intrafamilial variability in dystrophin abundance
    GENETICS

 correlated with a difference in the severity of the phenotype.

Duchenne muscular dystrophy
Becker dystrophy


Efficacy of spinal cord monitoring in neuromuscular scoliosis.

Somatosensory cortical evoked potentials (SCEPs) monitoring in neuromuscular scoliosis surgery was evaluated in a large consecutive series of spinal reconstructions to define its role in the detection and prevention of spinal cord injury; 141 procedures in 101 patients were evaluated. In 28% a reliable tracing could not be obtained. Two postoperative neurologic deficits were missed by SCEP monitoring. Diagnosis (Duchenne muscular dystrophy and polio) was the only significant factor found to correlate with a reliable tracing. The use of sublaminar wires was associated with a higher rate of change in tracing. Transient changes were common. SCEPs are unreliable and nonspecific in neuromuscular scoliosis surgery and are not efficatious in preventing or detecting spinal cord injury when used alone. Adjunctive techniques using epidural and MEPs must be studied in these patients.

    Somatosensory cortical evoked potentials (SCEPs) monitoring
    PARACLINICAL

 in

    neuromuscular scoliosis surgery
    MEDICATION

 was evaluated in a large consecutive series of

    spinal reconstructions
    MEDICATION

 to define its role in the detection and prevention of

    spinal cord injury
    SYMPTOMS

; 141 procedures in 101 patients were evaluated. In 28% a

    reliable tracing could not be obtained
    NEG_FINDINGS

. Two

    postoperative neurologic deficits
    SYMPTOMS

 were missed by

    SCEP monitoring
    PARACLINICAL

. Diagnosis (

    Duchenne muscular dystrophy
    DIAGNOSIS

 and

    polio
    DIAGNOSIS

) was the only significant factor found to correlate with a reliable tracing. The use of

    sublaminar wires
    MEDICATION

 was associated with a higher rate of

    change in tracing
    SYMPTOMS

.

    Transient changes
    SYMPTOMS

 were common.

    SCEPs
    PARACLINICAL

 are unreliable and nonspecific in

    neuromuscular scoliosis surgery
    MEDICATION

 and are not efficatious in preventing or detecting

    spinal cord injury
    SYMPTOMS

 when used alone.

    Adjunctive techniques using epidural and MEPs
    MEDICATION

 must be studied in these patients.

spinal cord injury
postoperative neurologic deficits
change in tracing
Transient changes
spinal cord injury


Myoblast transplantation between monozygotic twin girl carriers of Duchenne muscular dystrophy.

Monozygotic twin girls, both carriers of Duchenne muscular dystrophy, only one a severe symptomatic carrier and the other asymptomatic due to opposite lyonization, were studied. Myoblast clones were obtained from a muscle biopsy of the asymptomatic carrier. PCR amplification showed that most (94%) of these clones produced normal dystrophin mRNA. Roughly 704 million myoblasts were produced from 119 clones. These myoblasts were transplanted into the extensor carpi radialis (ECR) and in the biceps of one arm of the manifesting carrier while the other arm acted as the control. The strength of the patient was evaluated in a series of pre- and post-tests and a biopsy was obtained about 1 yr after the transplantation. The myoblast injections produced a significant force gain (12%-31%) in wrist extension but no force gain for elbow flexion. Muscle biopsies on the injected and control muscles obtained 1 yr after the injections showed only a small increase in the number of dystrophin positive fibers and the presence of numerous small type II fibers. The small beneficial effect of this transplantation cannot be attributed to immune problems, the donor and the recipient being identical twins, but may be due to a low level of spontaneous muscle regeneration.

    Monozygotic twin girls
    FAMILY

,

    both carriers of Duchenne muscular dystrophy
    FAMILY

,

    only one a severe symptomatic carrier and the other asymptomatic due to opposite lyonization
    FAMILY

, were studied. Myoblast clones were obtained from a

    muscle biopsy
    PARACLINICAL

 of the asymptomatic carrier.

    PCR amplification
    PARACLINICAL

 showed that most (94%) of these clones produced

    normal dystrophin mRNA
    GENETICS

. Roughly 704 million myoblasts were produced from 119 clones. These

    myoblasts were transplanted into the extensor carpi radialis (ECR) and in the biceps of
    MEDICATION

 one arm of the manifesting carrier while the other arm acted as the control. The strength of the patient was

    evaluated in a series of pre- and post-tests
    PARACLINICAL

 and a

    biopsy was obtained
    PARACLINICAL

 about 1 yr after the

    transplantation
    MEDICATION

. The

    myoblast injections
    MEDICATION

 produced a

    significant force gain (12%-31%) in wrist extension
    SYMPTOMS

 but

    no force gain for elbow flexion
    NEG_FINDINGS

.

    Muscle biopsies on the injected and control muscles
    PARACLINICAL

 obtained 1 yr after the injections showed

    only a small increase in the number of dystrophin positive fibers and the presence of numerous small type II fibers
    MICROSCOPIC

. The small beneficial effect of this

    transplantation
    MEDICATION

 cannot be attributed to

    immune problems
    NEG_FINDINGS

, the

    donor and the recipient being identical twins
    FAMILY

, but may be due to a

    low level of spontaneous muscle regeneration
    NEG_FINDINGS

.


Protein truncation test (PTT) to rapidly screen the DMD gene for translation terminating mutations.

We have developed a rapid and sensitive method to screen the Duchenne muscular dystrophy (DMD) mRNA for translation terminating mutations by a combination of RT-PCR (Reverse Transcription and Polymerase Chain Reaction) and in vitro transcription/translation applied to white blood cell mRNA. This technique was termed the protein truncation test (PTT). Here we demonstrate the detection of a point mutation in a DMD patient and his mother, a carrier. The PTT can also be used for carrier detection when no patient material is available, or in the case of spontaneous mutations. We developed a protocol to screen the total coding region of the DMD gene in 5-10 PTT reactions. Furthermore, PTT could be of diagnostic value in any disease where premature terminations form a substantial part of the total mutation spectrum.

We have developed a rapid and sensitive method to

    screen the Duchenne muscular dystrophy (DMD) mRNA
    PARACLINICAL

 for

    translation terminating mutations
    GENETICS

 by a combination of

    RT-PCR (Reverse Transcription and Polymerase Chain Reaction) and in vitro transcription/translation applied to white blood cell mRNA
    PARACLINICAL

. This technique was termed the

    protein truncation test (PTT)
    PARACLINICAL

. Here we demonstrate the detection of a

    point mutation
    GENETICS

 in a

    DMD
    DIAGNOSIS

 patient and

    his
    GENDER

    mother, a carrier
    FAMILY

. The PTT can also be used for carrier detection when no patient material is available, or in the case of

    spontaneous mutations
    GENETICS

. We developed a protocol to

    screen the total coding region of the DMD gene
    PARACLINICAL

 in

    5-10 PTT reactions
    PARACLINICAL

. Furthermore,

    PTT
    PARACLINICAL

 could be of diagnostic value in any disease where

    premature terminations
    GENETICS

 form a substantial part of the total mutation spectrum.

translation terminating mutations
point mutation
spontaneous mutations
premature terminations


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.

About two thirds of

    Duchenne muscular dystrophy (DMD)
    DIAGNOSIS

 patients have either

    gene deletions or duplications
    GENETICS

. The other

    DMD
    DIAGNOSIS

 cases are most likely the result of

    point mutations
    GENETICS

 that cannot be easily identified by current strategies. Utilizing a

    heteroduplex technique
    PARACLINICAL

 and

    direct sequencing of amplified products
    PARACLINICAL

, we screened our

    nondeletion/duplication DMD
    GENETICS

 population for

    point mutations
    GENETICS

. We now describe what we believe to be the first

    dystrophin missense mutation
    GENETICS

 in a

    DMD
    DIAGNOSIS

 patient. The

    mutation results in the substitution of an evolutionarily conserved leucine to arginine in the actin-binding domain
    GENETICS

. The patient makes a dystrophin protein which is properly localized and is present at a higher level than is observed in

    DMD
    DIAGNOSIS

 patients. This suggests that an intact actin-binding domain is necessary for protein stability and essential for function.

Duchenne muscular dystrophy (DMD)
DMD
DMD
DMD


Deficiency of dystrophin-associated proteins in Duchenne muscular dystrophy patients lacking COOH-terminal domains of dystrophin.

Dystrophin, the protein product of the Duchenne muscular dystrophy (DMD) gene, is a cytoskeletal protein tightly associated with a large oligomeric complex of sarcolemmal glycoproteins including dystroglycan, which provides a linkage to the extracellular matrix component, laminin. In DMD, the absence of dystrophin leads to a drastic reduction in all of the dystrophin-associated proteins, causing the disruption of the linkage between the subsarcolemmal cytoskeleton and the extracellular matrix which, in turn, may render muscle cells susceptible to necrosis. The COOH-terminal domains (cysteine-rich and carboxyl-terminal) of dystrophin have been suggested to interact with the sarcolemmal glycoprotein complex. However, truncated dystrophin lacking these domains was reported to be localized to the sarcolemma in four DMD patients recently. Here we report that all of the dystrophin-associated proteins are drastically reduced in the sarcolemma of three DMD patients in whom dystrophin lacking the COOH-terminal domains was properly localized to the sarcolemma. Our results indicate that the COOH-terminal domains of dystrophin are required for the proper interaction of dystrophin with the dystrophin-associated proteins and also support our hypothesis that the loss of the dystrophin-associated proteins in the sarcolemma leads to severe muscular dystrophy even when truncated dystrophin is present in the subsarcolemmal cytoskeleton.

Dystrophin, the protein product of the

    Duchenne muscular dystrophy (DMD) gene
    DIAGNOSIS

, is a cytoskeletal protein tightly associated with a large

    oligomeric complex of sarcolemmal glycoproteins including dystroglycan
    MICROSCOPIC

, which provides a linkage to the extracellular matrix component, laminin. In

    DMD
    DIAGNOSIS

, the

    absence of dystrophin
    BIOCHEMICAL

 leads to a

    drastic reduction in all of the dystrophin-associated proteins
    BIOCHEMICAL

, causing the

    disruption of the linkage between the subsarcolemmal cytoskeleton and the extracellular matrix
    SYMPTOMS

 which, in turn, may render

    muscle cells
    MICROSCOPIC

 susceptible to

    necrosis
    MICROSCOPIC

. The COOH-terminal domains

    (cysteine-rich and carboxyl-terminal) of dystrophin
    GENETICS

 have been suggested to interact with the sarcolemmal glycoprotein complex. However,

    truncated dystrophin lacking
    MICROSCOPIC

 these domains was reported to be

    localized to the sarcolemma
    MICROSCOPIC

 in four

    DMD
    DIAGNOSIS

 patients recently. Here we report that

    all of the dystrophin-associated proteins are drastically reduced in the sarcolemma
    MICROSCOPIC

 of three

    DMD
    DIAGNOSIS

 patients in whom

    dystrophin lacking the COOH-terminal domains was properly localized to the sarcolemma
    MICROSCOPIC

. Our results indicate that the COOH-terminal domains of dystrophin are required for the proper interaction of dystrophin with the dystrophin-associated proteins and also support our hypothesis that the

    loss of the dystrophin-associated proteins in the sarcolemma
    MICROSCOPIC

 leads to

    severe muscular dystrophy
    DIAGNOSIS

 even when truncated dystrophin is present in the

    subsarcolemmal cytoskeleton
    MICROSCOPIC

.

Duchenne muscular dystrophy (DMD) gene
DMD
DMD
DMD
severe muscular dystrophy


Immunohistological evidence for second or somatic mutations as the underlying cause of dystrophin expression by isolated fibres in Xp21 muscular dystrophy of Duchenne-type severity.

Using five monoclonal antibodies against different parts of the dystrophin molecule, we have studied the dystrophin composition of 17 dystrophin-positive fibres in a muscle biopsy from a boy with Xp21 muscular dystrophy of Duchenne-type severity. The fibres showed five distinct, reproducible, immunoreactive dystrophin profiles. All the profiles included both the N-terminal and the C-terminal domains, but between these domains, different fibres were negative for different antibodies, suggesting the somatic loss of certain exons. We interpret this as the first in situ evidence of an individual having different patterns of missing exons leading to restoration of the reading frame in various ways in the original germline frame-shifting deletion of exons 35-43. It follows that various somatic mutations had taken place in different fibres.

Using five monoclonal antibodies against different parts of the dystrophin molecule, we have studied the dystrophin composition of

    17 dystrophin-positive fibres
    MICROSCOPIC

 in a

    muscle biopsy
    PARACLINICAL

 from a

    boy
    GENDER

 with

    Xp21 muscular dystrophy of Duchenne-type severity
    DIAGNOSIS

. The fibres showed five distinct, reproducible,

    immunoreactive dystrophin profiles
    MICROSCOPIC

. All the profiles included both the

    N-terminal and the C-terminal
    BIOCHEMICAL

 domains, but between these domains, different fibres were

    negative for different antibodies
    NEG_FINDINGS

, suggesting the

    somatic loss of certain exons
    GENETICS

. We interpret this as the first in situ evidence of an individual having

    different patterns of missing exons leading to restoration of the reading frame in various ways in the original germline frame-shifting deletion of exons 35-43
    GENETICS

. It follows that various somatic mutations had taken place in different fibres.


An unusual case of Duchenne muscular dystrophy.

A 10-year-old boy with Duchenne muscular dystrophy (DMD), with quite unusual clinical data, is presented. He was unable to walk until age 6, walked only for 9 months and then became wheel-chair bound. No dystrophin was present on muscle biopsy sections and a large deletion was found in the dystrophin gene. The deletion encompassed the central high frequency deletion region of the gene. Early developmental milestones may be delayed in DMD, but patients usually start to walk around 2-3 years of age. A delay of the extent in this case is very unusual.

A

    10-year-old
    AGE

    boy
    GENDER

 with

    Duchenne muscular dystrophy (DMD)
    DIAGNOSIS

, with quite unusual clinical data, is presented.

    He
    GENDER

 was

    unable to walk until age 6
    NEG_FINDINGS

,

    walked only for 9 months
    NEG_FINDINGS

 and then became

    wheel-chair bound
    MEDICATION

.

    No dystrophin was present on muscle biopsy sections
    NEG_FINDINGS

 and a

    large deletion was found in the dystrophin gene
    GENETICS

. The

    deletion encompassed the central high frequency deletion region of the gene
    GENETICS

.

    Early developmental milestones
    SYMPTOMS

 may be delayed in

    DMD
    DIAGNOSIS

, but patients usually start to walk

    around 2-3 years of age
    AGE

. A

    delay of the extent
    AGE

 in this case is very unusual.


Mental retardation locus in Xp21 chromosome microdeletion.

Xp21 microdeletion syndrome is associated with variable size Xp21 deletions that usually include the glycerol kinase locus. The clinical phenotypes we studied in this chromosome region include: Xpter - Aland Island eye disease (AIED) -adrenal hypoplasia (AH) -glycerol kinase (GKD) -Duchenne muscular dystrophy (DMD) -retinitis pigmentosa (RP) -ornithine transcarbamylase (OTC) -centromere. In a compilation of 18 individuals in 14 families with the AH, GKD, and DMD loci deleted, 17 were male and all were developmentally delayed. In contrast, we report mentally retarded female carriers in two Xp21 deletion syndrome families with DMD, GKD, and AH in affected males. In the first family with normal karyotypes, a submicroscopic deletion was associated with DMD in the retarded male and with retardation in carrier females. In the second family an X chromosome with a cytogenetically deleted Xp21 distal to the OTC and RP genes segregated in the affected male and retarded female carriers. DNA analysis at the DMD locus verified the cytogenetic findings. This report of mental retardation in otherwise asymptomatic female carriers of Xp21 deletion classifies one form of mental retardation in females.

    Xp21 microdeletion syndrome
    DIAGNOSIS

 is associated with

    variable size Xp21 deletions that usually include the glycerol kinase locus
    GENETICS

. The clinical phenotypes we studied in this chromosome region include:

    Xpter - Aland Island eye disease (AIED) -adrenal hypoplasia (AH) -glycerol kinase (GKD) -Duchenne muscular dystrophy (DMD) -retinitis pigmentosa (RP)
    DIAGNOSIS

 -ornithine transcarbamylase (OTC) -centromere. In a compilation of 18 individuals in 14 families with the

    AH, GKD, and DMD loci deleted
    DIAGNOSIS

, 17 were

    male
    GENDER

 and all were

    developmentally delayed
    SYMPTOMS

. In contrast, we report

    mentally retarded
    SYMPTOMS

    female
    GENDER

 carriers in two

    Xp21 deletion syndrome
    DIAGNOSIS

 families with

    DMD
    DIAGNOSIS

,

    GKD
    DIAGNOSIS

, and

    AH
    DIAGNOSIS

 in affected

    males
    GENDER

. In the first family with

    normal karyotypes
    NEG_FINDINGS

, a

    submicroscopic deletion was associated with DMD
    GENETICS

 in the retarded

    male
    GENDER

 and with

    retardation
    SYMPTOMS

 in carrier

    females
    GENDER

. In the second family

    an X chromosome with a cytogenetically deleted Xp21 distal to the OTC and RP genes segregated in the affected male and retarded female carriers
    GENETICS

.

    DNA analysis at the DMD locus
    PARACLINICAL

 verified the

    cytogenetic findings
    PARACLINICAL

. This report of

    mental retardation
    SYMPTOMS

 in

    otherwise asymptomatic female
    NEG_FINDINGS

 carriers of

    Xp21 deletion
    GENETICS

 classifies one form of

    mental retardation
    DIAGNOSIS

 in

    females
    GENDER

.

developmentally delayed
mentally retarded
retardation
mental retardation

normal karyotypes
otherwise asymptomatic female


In utero fetal muscle biopsy for the diagnosis of Duchenne muscular dystrophy in a female fetus "suddenly at risk".

DNA methods to diagnose Duchenne muscular dystrophy (DMD) are not always informative, and we have published previously the first instance of in utero muscle biopsy to assess dystrophin in a male fetus having the same "X" as an affected sib. We present here a female fetus with a de novo X,1 translocation with breakpoint at Xp21, detected on amniocentesis for advanced maternal age. The translocation breakpoint placed her at high risk for DMD. In utero muscle biopsy at 20 weeks of gestation produced a specimen positive for dystrophin immunofluorescence indicating a likely normal fetus. The pregnancy was continued, and at term the baby girl was found to have normal serum creatine kinase levels, and was therefore unaffected with DMD. Our experiences add de novo Xp21 translocation to the indications for in utero muscle biopsy for diagnosis of DMD.

DNA methods to diagnose

    Duchenne muscular dystrophy (DMD)
    DIAGNOSIS

 are not always informative, and we have published previously the first instance of

    in utero muscle biopsy
    PARACLINICAL

 to

    assess dystrophin
    PARACLINICAL

 in a

    male
    GENDER

    fetus having the same "X" as an affected sib
    FAMILY

. We present here a

    female
    GENDER

    fetus
    AGE

 with a

    de novo X,1 translocation with breakpoint at Xp21
    GENETICS

, detected on

    amniocentesis
    PARACLINICAL

 for

    advanced maternal age
    AGE

. The

    translocation breakpoint
    GENETICS

 placed

    her
    GENDER

 at high

    risk for DMD
    FAMILY

.

    In utero muscle biopsy
    PARACLINICAL

 at

    20 weeks of gestation
    MEDICATION

 produced a

    specimen positive for dystrophin immunofluorescence
    BIOCHEMICAL

 indicating a

    likely normal fetus
    NEG_FINDINGS

. The

    pregnancy
    MEDICATION

 was continued, and at term the baby girl was found to have

    normal serum creatine kinase levels
    NEG_FINDINGS

, and was therefore

    unaffected with DMD
    NEG_FINDINGS

. Our experiences add

    de novo Xp21 translocation
    GENETICS

 to the indications for

    in utero muscle biopsy
    PARACLINICAL

 for diagnosis of

    DMD
    DIAGNOSIS

.

likely normal fetus
normal serum creatine kinase levels
unaffected with DMD


Insertion of a 5' truncated L1 element into the 3' end of exon 44 of the dystrophin gene resulted in skipping of the exon during splicing in a case of Duchenne muscular dystrophy.

We report here the second evidence of retrotransposition of L1, which was found inserted into the dystrophin gene of a patient, causing Duchenne muscular dystrophy (DMD). When the PCR was used to amplify a region of the dystrophin gene encompassing exon 44 from genomic DNA of two Japanese brothers with DMD, it was found to be approximately 600 bp larger than expected. Both the normal and the abnormally large products were amplified from the DNA of their mother. However, the maternal grandparents did not have the abnormal allele, and the mutation must therefore have occurred in the mother. Analysis of nucleotide sequence of the amplified product from a patient disclosed that the insertion was present zero to two bases upstream from the 3' end of exon 44 and that two to four bases of the exon sequence were deleted from the insertion site. The insertion sequence was found to be composed of 606-608 bp and to be almost identical to the inverse complement of 3' portion of the L1 retrotransposon consensus sequence. The dystrophin gene transcript from peripheral lymphocytes of one of the patients was analyzed by using reverse transcription/semi-nested PCR. The size of the amplified product encompassing exon 42 to 46 was smaller than expected. Sequencing of the amplified product disclosed that the sequence of exon 43 was directly joined to that of exon 45. Exon 44 of the transcript was thus shown to be skipped during splicing. This novel mutation of the dystrophin gene has important implications regarding retrotransposition of an active L1 element and provides a new insight into the origins of mutations in the dystrophin gene.

We report here the second evidence of

    retrotransposition of L1
    MICROSCOPIC

, which was found

    inserted into the dystrophin gene
    GENETICS

 of a patient, causing

    Duchenne muscular dystrophy (DMD)
    DIAGNOSIS

. When the

    PCR was used to amplify a region of the dystrophin gene encompassing exon 44 from genomic DNA
    PARACLINICAL

 of two

    Japanese
    ETHNICITY

    brothers
    GENDER

 with

    DMD
    DIAGNOSIS

, it was found to be

    approximately 600 bp larger than expected
    GENETICS

. Both the normal and the abnormally large products were amplified from the DNA of their mother. However,

    the maternal grandparents did not have the abnormal allele
    FAMILY

, and the

    mutation must therefore have occurred in the mother
    FAMILY

.

    Analysis of nucleotide sequence of the amplified product
    PARACLINICAL

 from a patient disclosed that

    the insertion was present zero to two bases upstream from the 3' end of exon 44 and that two to four bases of the exon sequence were deleted from the insertion site
    GENETICS

.

    The insertion sequence was found to be composed of 606-608 bp and to be almost identical to the inverse complement of 3' portion of the L1 retrotransposon consensus sequence
    GENETICS

.

    The dystrophin gene transcript from peripheral lymphocytes
    PARACLINICAL

 of one of the patients was

    analyzed by using reverse transcription/semi-nested PCR
    PARACLINICAL

. The

    size of the amplified product encompassing exon 42 to 46 was smaller than expected
    GENETICS

.

    Sequencing of the amplified product
    PARACLINICAL

 disclosed that

    the sequence of exon 43 was directly joined to that of exon 45. Exon 44 of the transcript was thus shown to be skipped during splicing
    GENETICS

. This

    novel mutation of the dystrophin gene
    GENETICS

 has important implications regarding

    retrotransposition of an active L1 element
    MICROSCOPIC

 and provides a new insight into the origins of

    mutations in the dystrophin gene
    GENETICS

.


Germinal mosaicism in a Duchenne muscular dystrophy family: implications for genetic counselling.

In this study we describe a three-generation family in which two siblings were affected by Duchenne muscular dystrophy (DMD). Immunohistochemical analysis of muscle dystrophin and haplotype analysis of the DMD locus revealed that the X chromosome carrying the DMD gene was transmitted from the healthy maternal grandfather to his three daughters, including the proband's mother. These findings indicate that the grandfather was a germinal mosaic for the DMD gene. The definition of the carrier status in two possible carriers led us to give accurate genetic counselling and to prevent the birth of an affected boy. The results of this study demonstrate the usefulness of haplotype analysis and immunohistochemical muscle dystrophin studies to detect hidden germinal mosaicism and to improve genetic counselling.

In this study we describe a

    three-generation family in which two siblings were affected by Duchenne muscular dystrophy (DMD)
    FAMILY

.

    Immunohistochemical analysis of muscle dystrophin
    PARACLINICAL

 and

    haplotype analysis of the DMD locus
    PARACLINICAL

 revealed that

    the X chromosome carrying the DMD gene was transmitted from the healthy maternal grandfather to his three daughters, including the proband's mother
    FAMILY

. These findings indicate that

    the grandfather was a germinal mosaic for the DMD gene
    FAMILY

. The definition of the carrier status in two possible carriers led us to give

    accurate genetic counselling
    MEDICATION

 and to prevent the birth of an affected

    boy
    GENDER

. The results of this study demonstrate the usefulness of

    haplotype analysis
    PARACLINICAL

 and

    immunohistochemical muscle dystrophin studies
    PARACLINICAL

 to detect

    hidden germinal mosaicism
    GENETICS

 and to improve

    genetic counselling
    MEDICATION

.

accurate genetic counselling
genetic counselling


A case of Duchenne muscular dystrophy with truncated dystrophin. Significance of a cysteine-rich domain for functional expression of dystrophin protein.

A Duchenne muscular dystrophy case showed truncated dystrophin (320 kDa) with an isoelectric point slightly shifted towards a more alkaline pH. From the polymerase chain reaction and immunochemical analysis data, the expressed dystrophin protein was predicted to lack the portion comprising the tail of the rod-like domain, the cysteine-rich domain, and the head of the C-terminal domain. These results indicated the functional importance of the cysteine-rich domain in the dystrophin protein.

A

    Duchenne muscular dystrophy
    DIAGNOSIS

 case showed

    truncated dystrophin (320 kDa)
    BIOCHEMICAL

 with an isoelectric point slightly shifted towards a more alkaline pH. From the

    polymerase chain reaction and immunochemical analysis data
    PARACLINICAL

, the expressed dystrophin protein was predicted to

    lack the portion comprising the tail of the rod-like domain, the cysteine-rich domain, and the head of the C-terminal domain
    GENETICS

. These results indicated the functional importance of the

    cysteine-rich domain in the dystrophin protein
    GENETICS

.


Dystrophin expression in Duchenne patients with "in-frame" gene deletions.

Details of disease progression and dystrophin expression are presented for three patients with Duchenne muscular dystrophy (DMD) who unexpectedly had intragenic deletions which maintained the open reading frame for mRNA translation. Analysis of dystrophin in muscle biopsies showed variable dystrophin synthesis in all three patients. Two with relatively small deletions (missing exons 10-13 and 52-55) had low levels of dystrophin which were comparable to those found in many DMD patients. The third patient (with a larger deletion which removed exons 3-25) produced dystrophin in the high abundance which is normally associated with patients who have Becker muscular dystrophy. This is the first time that a patient has been described with the clinical phenotype of DMD, a large amount of dystrophin which was correctly localized at the periphery of muscle fibres and an in-frame deletion of exons in the amino terminal domain.

Details of disease progression and dystrophin expression are presented for three patients with

    Duchenne muscular dystrophy (DMD)
    DIAGNOSIS

 who unexpectedly had

    intragenic deletions which maintained the open reading frame for mRNA translation
    GENETICS

.

    Analysis of dystrophin in muscle biopsies
    PARACLINICAL

 showed

    variable dystrophin synthesis
    BIOCHEMICAL

 in all three patients. Two with

    relatively small deletions (missing exons 10-13 and 52-55)
    GENETICS

 had

    low levels of dystrophin which were comparable to those found
    BIOCHEMICAL

 in many

    DMD
    DIAGNOSIS

 patients. The third patient (with a

    larger deletion which removed exons 3-25
    GENETICS

) produced

    dystrophin in the high abundance
    BIOCHEMICAL

 which is normally associated with patients who have

    Becker muscular dystrophy
    DIAGNOSIS

. This is the first time that a patient has been described with the clinical phenotype of

    DMD
    DIAGNOSIS

, a

    large amount of dystrophin which was correctly localized at the periphery of muscle fibres
    MICROSCOPIC

 and an

    in-frame deletion of exons in the amino terminal domain
    GENETICS

.

Duchenne muscular dystrophy (DMD)
DMD
Becker muscular dystrophy
DMD


[Manifesting carriers of Duchenne muscular dystrophy over two generations].

We report a family with manifesting DMD carriers over two generations. Sixty years old female (case 1) suffered from slowly progressive weakness since her thirties. Her youngest daughter aged 30 (case 2) had cramping calf muscle pain since her 5 years old. Progressive muscle weakness developed and lost her ambulation by the age of 20. Grandson of case 1 (son of case 1's eldest daughter who has no clinical symptoms) was diagnosed as DMD with deletion of exon 19-21 in dystrophin gene. Case 1 and case 2 were revealed to be DMD carriers. We speculate that, in this family, X-inactivation process was not random and paternal X was preferentially inactivated by maternal mutant X.

We report a

    family with manifesting DMD carriers over two generations
    FAMILY

.

    Sixty years old
    AGE

    female
    GENDER

 (case 1) suffered from

    slowly progressive weakness
    SYMPTOMS

    since her thirties
    AGE

.

    Her
    GENDER

    youngest daughter aged 30
    AGE

 (case 2) had

    cramping calf muscle pain
    SYMPTOMS

    since her 5 years old
    AGE

.

    Progressive muscle weakness
    SYMPTOMS

 developed and

    lost her ambulation
    SYMPTOMS

    by the age of 20
    AGE

.

    Grandson of case 1 (son of case 1's eldest daughter who has no clinical symptoms) was diagnosed as DMD
    FAMILY

 with

    deletion of exon 19-21 in dystrophin gene
    GENETICS

. Case 1 and case 2 were revealed to be

    DMD
    FAMILY

    carriers
    FAMILY

. We speculate that, in this family,

    X-inactivation process was not random
    GENETICS

 and

    paternal X was preferentially inactivated by maternal mutant X.
    GENETICS

slowly progressive weakness
cramping calf muscle pain
Progressive muscle weakness
lost her ambulation


Heterozygotic gene expression in endomyocardial biopsies: a new diagnostic tool confirms the Duchenne carrier status.

Identification of the defective gene and the absent gene product dystrophin can substantiate the clinical evidence for manifesting X-linked Duchenne type muscular dystrophy (DMD). It is not always possible, however, to rule out definitely a clinically asymptomatic carrier status in question, since even in the proven carrier DNA analysis is often inconclusive, and multinucleated skeletal muscle fibers express a basically normal membrane dystrophin. To substantiate the value of endomyocardial biopsy as a new tool for detection of the DMD carrier status we examined an endomyocardial biopsy of a volunteer who met the clinical criteria of a DMD carrier. Dystrophin immunohistochemistry and western blot of her skeletal muscle biopsy were inconclusive, and polymerase chain reaction and cDNA analysis failed to locate directly the X-chromosomal defect. We observed a clearcut mosaic of dystrophin-positive and -negative mononucleated cardiac muscle cells, reflecting a heterozygote carrier status in her endomyocardial biopsy, whereas 20 controls were uniformely positive. The incidence of DMD (1:3000 males) and especially the 30% spontaneous mutation rate, still the major pitfall in DNA analysis, show the need for an additional diagnostic tool.

Identification of the

    defective gene
    GENETICS

 and the

    absent gene product dystrophin
    BIOCHEMICAL

 can substantiate the clinical evidence for manifesting

    X-linked
    GENETICS

    Duchenne type muscular dystrophy (DMD)
    DIAGNOSIS

. It is not always possible, however, to rule out definitely a

    clinically asymptomatic
    NEG_FINDINGS

 carrier status in question, since even in the proven carrier

    DNA analysis is often inconclusive
    NEG_FINDINGS

, and

    multinucleated skeletal muscle fibers
    MICROSCOPIC

 express a

    basically normal membrane dystrophin
    NEG_FINDINGS

. To substantiate the value of

    endomyocardial biopsy
    PARACLINICAL

 as a new tool for detection of the

    DMD
    DIAGNOSIS

 carrier status we examined an

    endomyocardial biopsy
    PARACLINICAL

 of a

    volunteer
    GENDER

 who met the clinical criteria of a

    DMD
    DIAGNOSIS

 carrier.

    Dystrophin immunohistochemistry and western blot of her skeletal muscle biopsy were inconclusive
    NEG_FINDINGS

, and

    polymerase chain reaction and cDNA analysis
    PARACLINICAL

    failed to locate directly the X-chromosomal defect
    NEG_FINDINGS

. We observed a

    clearcut mosaic of dystrophin-positive and -negative mononucleated cardiac muscle cells
    MICROSCOPIC

, reflecting a

    heterozygote carrier status
    GENETICS

 in her

    endomyocardial biopsy
    PARACLINICAL

, whereas 20

    controls were uniformely positive
    NEG_FINDINGS

. The incidence of

    DMD
    DIAGNOSIS

 (1:3000 males) and especially the 30% spontaneous mutation rate, still the major pitfall in

    DNA analysis
    PARACLINICAL

, show the need for an additional diagnostic tool.

defective gene
X-linked
heterozygote carrier status

Duchenne type muscular dystrophy (DMD)
DMD
DMD
DMD


In situ hybridization shows direct evidence of skewed X inactivation in one of monozygotic twin females manifesting Duchenne muscular dystrophy.

A novel combination of conventional and molecular cytogenetic techniques was used to investigate the expression of an X-linked recessive disorder in one of monozygotic (MZ) twin females. These twins carry a deletion, approximately 300 kb in length, in one of their X chromosomes within the dystrophin gene, which is responsible for Duchenne muscular dystrophy (DMD) in one twin [Richards et al.: Am J Hum Genet 46:672-681, 1990]. A unique DNA fragment generated from an exon within this gene deletion was hybridized in situ to both twins' metaphase chromosomes, a probe which would presumably hybridize only to the normal X chromosome and not to the X chromosome carrying the gene deletion. Chromosomes were identified by reverse-banding (R-banding) and by the addition of 5-bromodeoxyuridine (BrdU) in culture to distinguish early and late replicating X chromosomes, corresponding to active and inactive X chromosomes, respectively. Hybridization experiments showed predominant inactivation of the normal X chromosome in the twin with DMD. This is the first report showing direct evidence at the chromosome level of unequal inactivation of cytogenetically normal X chromosomes resulting in the manifestation of an X-linked recessive disorder in one of monozygotic twin females. This study may now facilitate other research of unequal X inactivation and of females manifesting X-linked recessive disorders.

A novel combination of

    conventional and molecular cytogenetic techniques
    PARACLINICAL

 was used to investigate the expression of an

    X-linked recessive disorder
    GENETICS

 in one of

    monozygotic (MZ) twin females
    FAMILY

. These

    twins
    GENDER

 carry a

    deletion, approximately 300 kb in length, in one of their X chromosomes within the dystrophin gene, which is responsible for Duchenne muscular dystrophy (DMD)
    GENETICS

    in one twin
    FAMILY

 [Richards et al.: Am J Hum Genet 46:672-681, 1990]. A unique

    DNA fragment generated from an exon within this gene deletion
    PARACLINICAL

 was

    hybridized in situ to both twins' metaphase chromosomes
    PARACLINICAL

, a probe which would presumably hybridize only to the normal X chromosome and not to the

    X chromosome carrying the gene deletion
    GENETICS

. Chromosomes were identified by

    reverse-banding (R-banding)
    PARACLINICAL

 and by the addition of

    5-bromodeoxyuridine (BrdU) in culture
    PARACLINICAL

 to distinguish early and

    late replicating X chromosomes
    MICROSCOPIC

, corresponding to active and inactive

    X chromosomes
    GENETICS

, respectively.

    Hybridization experiments
    PARACLINICAL

 showed

    predominant inactivation of the normal X chromosome
    GENETICS

    in the twin with DMD
    FAMILY

. This is the first report showing direct evidence at the chromosome level of

    unequal inactivation of cytogenetically normal X chromosomes
    GENETICS

 resulting in the manifestation of an

    X-linked recessive disorder
    GENETICS

 in one of

    monozygotic twin females
    FAMILY

. This study may now facilitate other research of

    unequal X inactivation
    GENETICS

 and of

    females
    GENDER

 manifesting

    X-linked recessive disorders
    GENETICS

.


Dystrophin-positive myotubes in innervated muscle cultures from Duchenne and Becker muscular dystrophy patients.

Nerve-muscle co-cultures from five Duchenne muscular dystrophy (DMD) patients and one Becker (BMD) patient, were studied by immunocytochemistry with antibodies against different portions of dystrophin. Four DMD patients had a deletion in the dystrophin gene. Some dystrophin-positive myotubes were detected in a few samples of all DMD cases. PCR amplification of exon 8 of the dystrophin gene ruled out a contamination from rat spinal cord during innervation. Our results in three DMD cases, may be explained by a clonal selection of dystrophin-positive fibers observed in muscle biopsies, while in the other two cases, a "frame-restoring" mutation might account for the presence of dystrophin-positive myotubes. The possible expression of "dystrophin-related protein" or dystrophin immature isoform was considered. In the BMD case an abnormal truncated dystrophin was found in innervated muscle cultures, as well as in muscle biopsy.

    Nerve-muscle co-cultures
    MICROSCOPIC

 from five

    Duchenne muscular dystrophy (DMD)
    DIAGNOSIS

 patients and one

    Becker (BMD)
    DIAGNOSIS

 patient, were studied by

    immunocytochemistry
    PARACLINICAL

 with antibodies against different portions of dystrophin. Four

    DMD
    DIAGNOSIS

 patients had a

    deletion in the dystrophin gene
    GENETICS

.

    Some dystrophin-positive myotubes were detected in a few samples
    MICROSCOPIC

 of all

    DMD
    DIAGNOSIS

 cases.

    PCR amplification of exon 8 of the dystrophin gene
    PARACLINICAL

 ruled out a

    contamination from rat spinal cord during innervation
    NEG_FINDINGS

. Our results in three

    DMD
    DIAGNOSIS

 cases, may be explained by a

    clonal selection of dystrophin-positive fibers observed in muscle biopsies
    MICROSCOPIC

, while in the other two cases, a

    "frame-restoring" mutation
    GENETICS

 might account for the

    presence of dystrophin-positive myotubes
    MICROSCOPIC

. The possible expression of "

    dystrophin-related protein"
    BIOCHEMICAL

 or

    dystrophin immature isoform
    BIOCHEMICAL

 was considered. In the

    BMD
    DIAGNOSIS

 case an

    abnormal truncated dystrophin was found in innervated muscle cultures
    MICROSCOPIC

, as well

    as in muscle biopsy
    MICROSCOPIC

.

Duchenne muscular dystrophy (DMD)
Becker (BMD)
DMD
DMD
DMD
BMD

deletion in the dystrophin gene
"frame-restoring" mutation

dystrophin-related protein"
dystrophin immature isoform


Detection of gene deletions by PCR analysis in a Malaysian patient with Duchenne muscular dystrophy.

Duchenne muscular dystrophy (DMD), the commonest X-linked disorder, is a progressive, eventually fatal disease. With the advent of molecular genetics, the Duchenne gene and its protein product, dystrophin, have been characterised. Molecular diagnosis of DMD, identification of carriers and antenatal diagnosis are now possible. We describe here the use, in a Malaysian boy with DMD, of a recent innovation, multiplex polymerase chain reaction (PCR), to obtain molecular diagnosis by detection of dystrophin gene deletions.

    Duchenne muscular dystrophy (DMD)
    DIAGNOSIS

, the commonest

    X-linked disorder
    GENETICS

, is a progressive, eventually fatal disease. With the advent of

    molecular genetics
    PARACLINICAL

, the

    Duchenne gene
    GENETICS

 and its protein product, dystrophin, have been characterised.

    Molecular diagnosis
    PARACLINICAL

 of

    DMD
    DIAGNOSIS

, identification of carriers and

    antenatal diagnosis
    MEDICATION

 are now possible. We describe here the use, in a

    Malaysian
    ETHNICITY

    boy
    GENDER

 with

    DMD
    DIAGNOSIS

, of a recent innovation,

    multiplex polymerase chain reaction (PCR)
    PARACLINICAL

, to obtain

    molecular diagnosis
    PARACLINICAL

 by

    detection of dystrophin gene deletions
    PARACLINICAL

.

Duchenne muscular dystrophy (DMD)
DMD
DMD


Is the maintainance of the C-terminus domain of dystrophin enough to ensure a milder Becker muscular dystrophy phenotype?

The severe Duchenne muscular dystrophy (DMD) and the more benign Becker type (BMD) are allelic conditions, controlled by a defective gene at Xp21, caused by the absence (DMD) or a defect in quantity or quality (BMD) of the protein dystrophin. It has been suggested that the C-terminus domain of dystrophin is fundamental to ensure the proper protein sub-cellular localization and function. We wish to report our dystrophin findings in 4 among 142 DMD patients studied for DNA deletions and dystrophin analysis. Although they have a severe clinical course, a positive dystrophin immunofluorescence pattern was seen using C-terminal antibody, and a dystrophin band of reduced molecular weight (corresponding to their DNA deletions), but which maintained the C-terminus was seen through Western blot (WB). Based on these findings, we suggest that in order to partially maintain its function, resulting in a milder phenotype, dystrophin may carry large internal deletions but in addition to the C-terminus, the region encompassing both the N-terminus and the proximal region of the rod domain cannot be absent. Therefore, the prognosis of a Becker phenotype in a young patient should be done with caution if based only on the presence or not of dystrophin.

The

    severe Duchenne muscular dystrophy (DMD)
    DIAGNOSIS

 and the more

    benign Becker type (BMD)
    DIAGNOSIS

 are allelic conditions, controlled by a

    defective gene at Xp21
    GENETICS

, caused by the

    absence
    BIOCHEMICAL

 (

    DMD
    DIAGNOSIS

) or a

    defect in quantity or quality
    BIOCHEMICAL

 (

    BMD
    DIAGNOSIS

)

    of the protein dystrophin
    BIOCHEMICAL

. It has been suggested that the C-terminus domain of dystrophin is fundamental to ensure the proper protein sub-cellular localization and function. We wish to report our dystrophin findings in 4 among 142

    DMD
    DIAGNOSIS

 patients studied for

    DNA deletions
    PARACLINICAL

 and

    dystrophin analysis
    PARACLINICAL

. Although they have a

    severe clinical course
    SYMPTOMS

, a

    positive dystrophin immunofluorescence
    BIOCHEMICAL

 pattern was seen using

    C-terminal antibody
    BIOCHEMICAL

, and a

    dystrophin band of reduced molecular weight (corresponding to their DNA deletions
    SYMPTOMS

), but which

    maintained the C-terminus
    BIOCHEMICAL

 was seen through

    Western blot (WB)
    PARACLINICAL

. Based on these findings, we suggest that in order to partially maintain its function, resulting in a milder phenotype, dystrophin may carry

    large internal deletions but in addition to the C-terminus, the region encompassing both the N-terminus and the proximal region of the rod domain cannot be absent
    GENETICS

. Therefore, the prognosis of a

    Becker
    DIAGNOSIS

 phenotype in a

    young
    AGE

 patient should be done with caution if based only on the presence or not of dystrophin.

absence
defect in quantity or quality
of the protein dystrophin
positive dystrophin immunofluorescence
C-terminal antibody
maintained the C-terminus


Diagnosis of dystrophinopathies: review for the clinician.

The dystrophinopathies are muscle disorders due to an abnormality of an Xp21-linked gene which produces the dystrophin protein. The most common of these disorders are the Duchenne and Becker muscular dystrophies. Modern molecular genetic techniques enable reliable diagnosis and prognosis in many patients, but there are occasional pitfalls. Furthermore, the clinical spectrum of the dystrophinopathies are now such that the clinician needs to be aware of a broader range of clinical disorders that require analysis of the dystrophin gene and its product, not just those that mirror a classic Duchenne or Becker muscular dystrophy picture. This spectrum ranges from a severe form presenting at birth to asymptomatic elevation of CK. Females may be manifesting carriers or present as a severe phenotype when the abnormal gene is expressed as an X-autosome translocation or monosomy X. Laboratory diagnosis and prognosis can be made most accurately by using both DNA analysis at the dystrophin gene and immuno-analysis of muscle with antibodies directed against different regions of the protein product. This review describes some exemplary patients, suggests a clinical classification for dystrophinopathies, and outlines a diagnostic approach.

The

    dystrophinopathies
    DIAGNOSIS

 are

    muscle disorders
    DIAGNOSIS

 due to an

    abnormality of an Xp21-linked gene which produces the dystrophin protein
    GENETICS

. The most common of these disorders are the

    Duchenne and Becker muscular dystrophies
    DIAGNOSIS

. Modern molecular genetic techniques enable reliable diagnosis and prognosis in many patients, but there are occasional pitfalls. Furthermore, the clinical spectrum of the

    dystrophinopathies
    DIAGNOSIS

 are now such that the clinician needs to be aware of a broader range of clinical disorders that require

    analysis of the dystrophin gene and its product
    PARACLINICAL

, not just those that mirror a classic

    Duchenne or Becker muscular dystrophy
    DIAGNOSIS

 picture. This spectrum ranges from a severe form presenting at

    birth
    AGE

 to

    asymptomatic elevation of CK
    BIOCHEMICAL

.

    Females
    GENDER

 may be manifesting carriers or present as a severe phenotype when the abnormal gene is expressed as an X-autosome translocation or monosomy X.

    Laboratory diagnosis
    PARACLINICAL

 and prognosis can be made most accurately by using both

    DNA analysis at the dystrophin gene
    PARACLINICAL

 and

    immuno-analysis of muscle
    PARACLINICAL

 with antibodies directed against different regions

    of the protein product
    PARACLINICAL

. This review describes some exemplary patients, suggests a clinical classification for

    dystrophinopathies
    DIAGNOSIS

, and outlines a diagnostic approach.


The use of monoclonal antibodies in diagnostic tests for Becker and Duchenne muscular dystrophy.

Monoclonal antibodies recognizing different epitopes of dystrophin have now been widely applied in diagnostic tests for Duchenne and Becker muscular dystrophy (BMD). The preservation of the C-terminus in BMD patients necessitates the routine use of one antibody raised against this region. Additional antibodies against deletion-prone regions of dystrophin further increase the diagnostic power of the tests. We describe two cases which illustrate that the combined use of different antibodies is necessary to avoid misdiagnoses of BMD at the protein level.

Monoclonal antibodies recognizing different epitopes of dystrophin have now been widely applied in diagnostic tests for

    Duchenne
    DIAGNOSIS

 and

    Becker muscular dystrophy (BMD)
    DIAGNOSIS

. The preservation of the C-terminus in

    BMD
    DIAGNOSIS

 patients necessitates the routine use of one antibody raised against this region. Additional antibodies against

    deletion-prone regions of dystrophin
    GENETICS

 further increase the diagnostic power of the tests. We describe two cases which illustrate that the combined use of different antibodies is necessary to avoid misdiagnoses of

    BMD
    DIAGNOSIS

 at the protein level.

Duchenne
Becker muscular dystrophy (BMD)
BMD
BMD


Exon 44 nonsense mutation in two-Duchenne muscular dystrophy brothers detected by heteroduplex analysis.

Utilizing a heteroduplex method, we screened the dystrophin exon 43-45 region for point mutations, including small deletions and insertions. The method depends upon the formation of a heteroduplex between wild-type and mutant DNA PCR products. DNA specimens from one hundred and four DMD patients without detected deletions or duplications were multiplexed amplified for exons 43, 44, and 45. The PCR products were mixed with the PCR products from nonaffected controls, electrophoresed, and examined for the presence of altered mobility heteroduplex bands. An exon 44 nonsense mutation in two DMD brothers and a common intron 44 polymorphism were identified using this approach. Although the exon 44-45 region is a hotspot for deletion breakpoints, it does not appear to be prone to point mutations. The technique is extremely useful for screening several exons simultaneously and it allowed us to screen a large number of patients.

Utilizing a

    heteroduplex method
    PARACLINICAL

, we

    screened the dystrophin exon 43-45 region
    PARACLINICAL

 for

    point mutations
    GENETICS

, including

    small deletions and insertions
    GENETICS

. The method depends upon the formation of a heteroduplex between wild-type and mutant DNA PCR products.

    DNA specimens
    PARACLINICAL

 from one hundred and four

    DMD
    DIAGNOSIS

 patients

    without detected deletions or duplications
    NEG_FINDINGS

 were

    multiplexed amplified for exons 43, 44, and 45
    PARACLINICAL

. The PCR products were mixed with the PCR products from nonaffected controls, electrophoresed, and examined for the presence of

    altered mobility heteroduplex bands
    SYMPTOMS

. An

    exon 44 nonsense mutation in two DMD brothers
    GENETICS

 and a

    common intron 44 polymorphism
    GENETICS

 were identified using this approach. Although

    the exon 44-45 region is a hotspot for deletion breakpoints
    GENETICS

, it does not appear to be prone to

    point mutations
    GENETICS

. The technique is extremely useful for screening several exons simultaneously and it allowed us to screen a large number of patients.


Variability in clinical, genetic and protein abnormalities in manifesting carriers of Duchenne and Becker muscular dystrophy.

We have analysed the results of clinical assessment, X-inactivation status, deletion screening and dystrophin analysis in eight manifesting carriers of Duchenne and Becker muscular dystrophy (DMD and BMD). Only two had a prior family history of X-linked muscle disease, all had normal karyotypes and none were twins. Presentation varied from 2 to 25 yr and progression varied from a DMD-like severity to a very mild BMD-like course. In one girl the initial symptoms were restricted to learning difficulties. Where methods for assessing X-inactivation were informative, three patients showed an abnormal pattern. However, in one patient, the obligate carrier daughter of a BMD patient who had presented at the age of 2 yr, X-inactivation appeared normal in lymphocytes and muscle. While dystrophin analysis seems to be reliable in identifying manifesting carriers of DMD and BMD, the relationship between X-inactivation status, dystrophin analysis and phenotype is not simple.

We have analysed the results of clinical assessment,

    X-inactivation status
    PARACLINICAL

,

    deletion screening
    PARACLINICAL

 and

    dystrophin analysis
    PARACLINICAL

 in eight manifesting carriers of

    Duchenne and Becker muscular dystrophy (DMD and BMD)
    DIAGNOSIS

. Only two had

    a prior family history of X-linked muscle disease
    FAMILY

, all had

    normal karyotypes
    NEG_FINDINGS

 and

    none were twins
    FAMILY

. Presentation varied from

    2 to 25 yr
    AGE

 and progression varied from

    a DMD-like severity to a very mild BMD-like course
    SYMPTOMS

. In one

    girl
    GENDER

 the initial symptoms were

    restricted to learning difficulties
    SYMPTOMS

. Where methods

    for assessing X-inactivation
    PARACLINICAL

 were informative, three patients showed an abnormal pattern. However, in one patient,

    the obligate carrier daughter of a BMD patient who had presented at the age of 2 yr
    FAMILY

,

    X-inactivation appeared normal in lymphocytes and muscle
    NEG_FINDINGS

. While

    dystrophin analysis
    PARACLINICAL

 seems to be reliable in identifying manifesting carriers of

    DMD
    DIAGNOSIS

 and

    BMD
    DIAGNOSIS

, the relationship between

    X-inactivation status
    PARACLINICAL

,

    dystrophin analysis
    PARACLINICAL

 and phenotype is not simple.


Diagnosis of DMD carrier status in a family with no known affected males.

A 30-year-old woman and her two-year-old daughter were found by chance to have moderately raised serum creatine kinase (CK) levels. Since the mother was pregnant, the authors investigated the possibility that the two females were carriers of the common Duchenne muscular dystrophy (DMD) gene. No immunohistochemical abnormality was detected in the mother, but in the daughter a clear mosaic pattern of dystrophin positive and negative fibres was found, indicating carrier status for DMD. These data indicate that a diagnosis of DMD carrier status can be made even in families without a positive history for this disorder; therefore, immunocytochemical studies, using antidystrophin antibodies, should be performed on all females with raised CK levels, including the youngest.

A

    30-year-old
    AGE

    woman
    GENDER

 and

    her
    GENDER

    two-year-old
    AGE

 daughter were found by chance to have

    moderately raised serum creatine kinase (CK) levels
    BIOCHEMICAL

. Since the

    mother
    GENDER

 was

    pregnant
    GENDER

, the authors investigated the possibility that the two

    females
    GENDER

 were

    carriers of the common Duchenne muscular dystrophy (DMD) gene
    GENETICS

.

    No immunohistochemical abnormality was detected in the mother
    NEG_FINDINGS

, but in the daughter a

    clear mosaic pattern of dystrophin positive and negative fibres
    MICROSCOPIC

 was found, indicating

    carrier status for DMD
    GENETICS

. These data indicate that a diagnosis of

    DMD
    DIAGNOSIS

 carrier status can be made even in

    families without a positive history for this disorder
    FAMILY

; therefore,

    immunocytochemical studies
    PARACLINICAL

, using

    antidystrophin antibodies
    PARACLINICAL

, should be performed on all

    females
    GENDER

 with

    raised CK levels
    BIOCHEMICAL

, including the

    youngest
    AGE

.


Female twin with Hunter disease due to nonrandom inactivation of the X-chromosome: a consequence of twinning.

We report the occurrence of Hunter disease (mucopolysaccharidosis type II) in a karyotypically normal girl who was one of identical twins. Molecular studies showed nonrandom X-inactivation in both her fibroblasts and lymphocytes, while her normal twin showed equal usage of both X chromosomes. In view of previous reports of 7 pairs of identical female twins in which one had Duchenne muscular dystrophy, it seems that twinning may be strongly associated with nonrandom X-inactivation, and is not specific to the properties of the disease causing gene.

We report the occurrence of

    Hunter disease (mucopolysaccharidosis type II)
    DIAGNOSIS

 in a

    karyotypically normal
    NEG_FINDINGS

    girl
    GENDER

    who was one of identical twins
    FAMILY

.

    Molecular studies
    PARACLINICAL

 showed

    nonrandom X-inactivation in both her fibroblasts and lymphocytes
    GENETICS

, while

    her
    GENDER

    normal twin showed equal usage of both X chromosomes
    FAMILY

. In view of previous reports of

    7 pairs of identical female twins in which one had Duchenne muscular dystrophy
    FAMILY

, it seems that

    twinning
    FAMILY

 may be strongly associated with

    nonrandom X-inactivation
    GENETICS

, and is not specific to the properties of the disease causing gene.