Detection of carriers of Duchenne and Becker muscular dystrophy using the fluorescence in situ hybridization method

Duchenne and Becker muscular dystrophies (DMD/BMD) are X-linked recessive disorders caused by mutations in the dystrophin gene. A large intragenic deletion has been described in about 65% of DMD/BMD patients. Mothers of affected males are DMD/BMD carriers in two thirds of the cases. Routine deletions detection in DMD/BMD males is performed using multiplex polymerase chain reaction (mPCR), RT-PCR with a protein truncation test (PTT) or using Southern blotting. In females the deletions detection is complicated by the presence of a normal gene copy on the second X-chromosome. We are presenting the diagnostic strategy using FISH for the deletions detection in the dystrophin gene of female DMD/BMD carriers. We have used a set of six cosmid probes for the detection of the most frequently deleted areas of the dystrophin gene from the Department of Human Genetics, Leiden University Medical Center. We have examined 14 mothers of DMD/BMD males with a deletion in the dystrophin gene identified using mPCR. Four mothers of affected males have been diagnosed as carriers of a deletion in the dystrophin gene. We have revealed no deletion mutations in the exons examined in a control group of four healthy females. No discrepancy has been found between the FISH analysis results and the results of mPCR. Our results indicate that FISH is an effective and direct method for the identification of DMD/BMD carriers and we suggest this method as a method of a first choice in the identification of DMD/BMD carriers.     

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 – Åland 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. © 1993 Wiley-Liss, Inc.     

Identification of female carriers for Duchenne and Becker muscular dystrophies using a FISH-based approach

Duchenne muscular dystrophy (DMD) and Becker muscular dystrophy (BMD) are X-linked recessive neuromuscular diseases caused by dystrophin gene mutations. Deletions, or more rarely duplications, of single or multiple exons within the dystrophin gene can be detected by current molecular methods in approximately 65% of DMD patients. Mothers of affected males have a two-thirds chance of carrying a dystrophin mutation, whilst approximately one-third of affected males have de novo mutations. Currently, Southern blot analysis and multiplex PCR directed against exons in deletion hot spots are used to determine female carrier status. However, both of these assays depend on dosage assessment to accurately identify carriers since, in females, the normal X chromosome is also present. To obviate quantitation of gene dosage, we have developed exon-specific probes from the dystrophin gene and applied them to a screen for potential carrier females using fluorescence in situ hybridization (FISH). Cosmid clones, representing 16 exons, were identified and used in FISH analysis of DMD/BMD families. Our preliminary work has identified multiple, informative probes for several families with dystrophin deletions and has shown that a FISH-based assay can be an effective and direct method for establishing the DMD/BMD carrier status of females.     

Duchenne肌营养不良症患者及携带者的基因缺失和重复突变检测

目的利用多重连接依赖探针PCR扩增技术检测Duchenne肌营养不良症（Duchenne muscular dystrophy，DMD）患者及其可能的女性携带者的dystrophin基因的缺失、重复突变。方法利用多重连接依赖探针PCR扩增对32例DMD患者及其27个可能的女性携带者的dystrophin基因缺失、重复进行检测。结果32个先证者中，共检测出了24例DMI）患者具有一个或多个外显子的缺失，l例DMD患者具有重复突变，l例患者为第19外显子的无义突变（R768X），6例没有检测出缺失、重复突变的先证者可能是点突变所致。17个先证者的18位女性亲属具有和先证者相同的缺失、重复突变。结论多重连接依赖探针PCR扩增技术可用于检测DMD基因的缺失、重复突变，可以检测DMD基因女性携带者的基因杂合情况，在检测DMD基因缺失和重复方面，具有一定的应用价值。     

Arylamine N-acetyltransferase type 2 (NAT2), chromosome 8 aneuploidy, and identification of a novel NAT1 cosmid clone: an investigation in bladder cancer by interphase FISH.

Two genes (arylamine N-acetyltransferase types 1 and 2, NAT1 and NAT2), which are known to metabolize bladder carcinogens, are located on chromosome band 8p22. Alterations in chromosome 8, including deletions of 8p, occur frequently in many epithelium-derived tumors. In this study, fluorescence in situ hybridization (FISH) was used for study of the relationship between chromosome 8 deletions in the region of NAT1 and NAT2 and grade and stage of tumor in bladder cancer. Cells from 52 bladder tumors were examined by dual-labeling FISH with a centromere 8-specific probe and a cosmid probe for NAT2. A more limited number were examined for loss with both the NAT2 probe and a newly constructed NAT1-specific cosmid. Loss of NAT2 was found in 6/52 patients in more than 30% of cells, and in 10/52 in 10%-30% of cells examined. Six samples also showed loss of NAT1, indicating that the region of deletion spans at least the distance of the two genes. No obvious correlation between loss of NAT genes with grade and stage of tumor was evident. Interestingly, 17/52 (32%) tumors showed an increased copy number of chromosome 8, with tumors of low stage showing relatively smaller increases of chromosome 8. Loss of 8p22 and genetic instability involving chromosome 8 indicate that this chromosome is important in bladder cancer and that NAT genes will act as important genetic landmarks in defining deletions in this disease. Genes Chromosomes Cancer 25:376-383, 1999.     

Fluorescence in situ hybridization for detecting TP16 MTS1/CDK41 gene deletions in squamous cell carcinoma of the head and neck

We have previously shown TP16 MTS1/CDK41 gene deletion in more than 50% of a cohort of squamous cell carcinoma of the head and neck (SCCHN) patients using polymerase chain reaction (PCR). We have performed fluorescence in situ hybridization (FISH) on paraffin-embedded SCCHN specimens from the same cohort to identify the deletion of TP16 MTS1/CDK41CDK41gene. Twenty normal and 19 SCCHN specimens were studied. An alpha-satellite DNA probe specific for chromosome 9 and a cosmid probe for the TP16 MTS1/CDK41CDK41gene were used. Of the 19 tumors examined by FISH, 6 had homozygous deletions, 7 were hemizygously deleted, and the remaining 6 showed no evidence of deletion of the TP16 MTS1/CDK41 gene. None of the normal specimens showed TP16 gene deletion. Data obtained from FISH highly correlated with the PCR results for the identification of TP16 MTS1/CDK41 gene deletions. Patients with deletion of the TP16 MTS1/CDK41 gene show a greater tendency toward the development of recurrence and metastasis.     

Molecular cytogenetic studies of duplication 9q32→q34.3 inserted into 9q13

Fluorescence in situ hybridization (FISH) studies using whole chromosome 9 painting probe, classical satellite (9q12-specific) probe and abl cosmid probe (locus: 9q34) were performed on a female infant who was born with multiple congenital anomalies and the karyotype 46,XX, 9q+. The results of FISH confirm the euchromatic nature of the extra material on the long arm of chromosome 9, and provide evidence that it is of chromosome 9 origin. The structural rearrangement has probably resulted from an insertion of a duplicated segment 9q32→q34.3 into band q13, as shown by the abl cosmid probe. The clinical features in this patient are similar to the previously reported cases of partial trisomy 9q3.     

间期FISH分析中细胞类型及探针的选择

荧光原位杂交已成为医学遗传学领域中一项极为重要的研究手段。为了摸索不同来源的产前诊断材料的间期核进行荧光原位杂交的可行性，选择了α重复序列ＤＮＡ及ＣｏｓｍｉｄＤＮＡ为探针，分别与未培养的绒毛间质细胞、羊水细胞及外周血白细胞的间期核杂交。结果表明：各种材料的间期核均见杂交信号，１３／２１α重复序列探针用于未培养的绒毛间质细胞及外周血白细胞的杂交信号最为特异，能有效的检出染色体非整倍体，羊水细胞则远不如绒毛细胞，绒毛组织是最理想的产前诊断材料。     

Mutation analysis and prenatal diagnosis for 50 pedigrees affected with Duchenne/Becker muscular dystrophyCSCD

Objective To establish individualized prenatal diagnosis program for families affected with Duchenne/Becker muscular dystrophy (DMD/BMD) and different clinical background using a variety of methods. Methods Multiplex ligation-dependent probe amplification (MLPA) was performed on 50 patients suspected for DMD/BMD. For single exon deletions of the DMD gene, PCR was used for validating the results. For those without any deletion or duplication, Sanger sequencing was used to screen for DMD gene mutations in the children and their mothers. Prenatal genetic testing was provided to female carriers using chorionic villus, amniocentesis or cord blood samples. To ensure the accuracy of diagnosis, all prenatal specimens were also subjected to linkage analysis. Results Among the 50 patients with DMD/ BMD, 23 harbored large deletions, 11 only had single exon deletions, 10 harbored duplications, and 5 had small scare mutations. No mutation was detected in one family. For 37 women undergoing prenatal diagnosis, 10 fetuses were identified as affected males, 6 were female carriers, while 21 were not found to carry any mutation. Testing of creatine kinase was consistent with the results of prenatal diagnosis. For a patient harboring exon 51 deletion, the same mutation was found in a fetus but not in their mother. The proband and fetus had inherited the same haplotype, which suggested that the mother probably has germline mosaicism for the mutation. Conclusion Application of individualized methods for analyzing pregnant women with different clinical background can minimize the risk for giving birth to further children affected with DMD/BMD. © 2018 West China University of Medical Sciences. All rights reserved.     

Prenatal in situ hybridization test for deleted steroid sulfatase gene

X-linked ichthyosis results from steroid sulfatase (STS) deficiency; 90% of affected patients have a complete deletion of the entire 146 kb STS gene on the distal X chromosome short arm (Xp22.3). In these families prenatal diagnosis and carrier testing can be completed in 2 days by hybridizing simultaneously 2 different cosmid probes labeled with fluorescein or Texas red and counterstaining interphase nuclear DNA with DAPI. An STS gene probe labeled with Texas red hybridizes specially to the steroid sulfatase gene on the X chromosome. A second flanking probe labeled with fluorescein hybridizes to both the normal Y chromosome and normal and STS deleted X chromosomes. In this fashion the interphase nuclei of normal males, affected males, normal females, and carrier females can be distinguished unambiguously. Because normal males and carrier females each show two yellow-green fluorescein spots and one Texas red STS spot, use of this test prenatally requires determining fetal sex independently with repetitive X and Y chromosome specific probes. This procedure can be used with lymphocytes, direct and cultured chorionic villus cells, direct and cultured amniocytes, and fibroblasts. Similar methods are anticipated to be useful for rapid diagnostic assessment of other aneuploid gene disorders. © 1993 Wiley-Liss, Inc.     

Duchenne型肌营养不良症家系的多重PCR和STR—PCR基因诊断

目的该研究率先开展温州地区Duchenne型肌营养不良症（DMD）家系的缺失基因诊断特别是STR单体型连锁基因诊断,为基于DMD症状前、携带者基因诊断结果的遗传咨询和生育指导提供依据。方法针对4例DMD先证者,采用多重PCR检测常见18个外显子缺失,进行直接基因诊断。针对未能发现常见外显子缺失的DMD先证者及其有关家系成员,采用短串联重复序列（STR）PCR检测5个位点（3’CA、44CA、45CA、49CA和50CA）STR多态性,进行间接单体型连锁基因诊断。结果家系二的先证者缺失外显子3、4和6。其余3个家系的先证者的异常x染色体均肯定来源于其母亲。家系一先证者外婆肯定是携带者。家系三先证者年幼（4周岁）弟弟肯定为正常人,将来年龄大了也不会发病,先证者外婆肯定是携带者。家系四先证者刚出生的妹妹肯定是遗传携带者,将来其生育儿子有遗传患病风险。结论该研究的DMD家系的缺失和STR单体型连锁基因诊断,特别是对症状前男孩的诊断、对无患病后代的女性携带者的检出,具有非常重要的实际意义,可以为遗传咨询和生育指导提供可靠依据。     

Fabry disease: Comparison of enzymatic,linkage, and mutation analysis for carrier detection in a family with a novel mutation (30delG)

Fabry disease (FD) is an X-linked recessive disorder caused by the deficient activity of the lysosomal enzyme α-galactosidase A (α-Gal A). Affected males are reliably diagnosed by demonstration of deficient α-Gal A activity in plasma or leukocytes. However, identification of female carriers is problematic due to Lyonization, requiring mutation identification and/or linkage studies for accurate carrier detection. Here, we describe a large Brazilian kindred with Fabry disease that permitted comparison of biochemical and molecular diagnostic techniques. Initially, the plasma α-Gal A activities were determined in at-risk affected males and potential female carriers; affected males were readily diagnosed, while the females had variable results. To detect carrier females, haplotype analysis using 10 polymorphic markers adjacent to the gene was performed. Subsequently, solid-phase direct sequencing of the α-Gal A gene demonstrated a novel single base deletion in exon 1 (30delG). Discrepancies were observed between the enzymatic and molecular diagnoses in two at-risk females. These findings emphasize the need for precise heterozygote diagnosis by mutation and/or haplotype analyses in all families with Fabry disease. Am. J. Med. Genet. 84:420–424, 1999. © 1999 Wiley-Liss, Inc.     

Analysis of mutations at the fragile X locus using the DNA probe Ox1.9.

In this study, 40 families segregating for fragile X [fra (X)] syndrome were examined for the presence of a mutation within the FMR-1 gene. Using the DNA probe Ox1.9, both carriers and affected individuals were found to contain an insertion/amplification-type of mutation with somatic instability. Variability in the size of the mutation, which ranged from less than 0.2 kb to approximately 13 kb, was observed both between individuals (even from the same family) and within individuals, who showed a smear rather than a discrete band(s) on Southern blot analysis. Transmission of the mutation by males resulted in little change of its size, while transmission by females usually resulted in an increase in size. Correlations were observed between the size of inserted/amplified DNA and the level of chromosome fragility and the presence or absence of mental impairment. Overall, a mutation was detected in 66 of 67 (99%) clinically affected males, in 12 of 13 (92%) transmitting males and in 95 of 112 (85%) carrier females. Equivocal results were obtained in 12 (11%) of the carrier females. No mutation was detected in 58 females and 33 males predicted to be normal by linkage, or in one female and 36 normal control males. These results strongly suggest that the mutation detected by Ox1.9 is closely associated with the cytogenetic and clinical expression of fra (X) syndrome. Additionally, the use of this probe along with other probe/enzyme combinations should provide a sensitive clinical assay for the detection of carriers of fra (X) syndrome.     

Supernumerary ring chromosome 20 characterized by fluorescence in situ hybridization

We report on a boy with mild dysmorphic features and developmental delay, in whom karyotyping showed an additional minute ring chromosome in 60% of metaphases. Fluorescence in situ hybridization (FISH) with a centromere specific probe demonstrated that the ring chromosome contained the centromeric region of chromosome 20. The ring was highlighted completely using a chromosome 20 painting probe. A cosmid probe for 20p12-13 gave a positive signal and hybridization with an all-telomere probe showed one signal, suggesting a breakpoint in the 20p telomere. The results suggested that only a small part of 20q was involved in this ring. The ring was also detected in 18% of nuclei of a buccal smear. The phenotypic similarities of symptoms in the proband to patients with a (partial) trisomy 20p and the dissimilarities to symptoms in patients with (partial) trisomy 20q were in agreement with the FISH results.     

Combined use of PRINS and FISH in the study of the dystrophin gene.

The efficacy of fluorescence in situ hybridization (FISH) may be limited in specific applications by low-resolution sensitivity. Primed in situ labeling (PRINS) is based on specific hybridization of an unlabeled oligonucleotide with a denatured template and synthesis of a single-strand DNA in situ. This method may represent a powerful alternative to FISH for gene mapping because of its ability to generate multiple independent signals within the same gene segment. We investigated the specificity of signals produced by a modified PRINS protocol combining a centromeric probe for the X-chromosome with specific primers for 3'- and 5'-terminal regions of the dystrophin gene. In approximately 70% of nuclei from male and female subjects, we detected one or two large signals (X-chromosome centromere) and two or four smaller signals (the two regions of the dystrophin gene). Specific hybridization of the oligonucleotides on Xp was demonstrated by localization of the smaller (dystrophin) and larger (X-centromere) signals on the same chromosome. Simultaneous hybridization with a centromeric probe and gene-specific oligonucleotides allowed localization of PRINS signals, and assessment of the specificity of the primers used for hybridization. This approach could facilitate identification of female carriers of small intragenic deletions in the dystrophin gene.     

Combined use of PRINS and FISH in the study of the dystrophin gene

The efficacy of fluorescence in situ hybridization (FISH) may be limited in specific applications by low‐resolution sensitivity. Primed in situ labeling (PRINS) is based on specific hybridization of an unlabeled oligonucleotide with a denatured template and synthesis of a single‐strand DNA in situ. This method may represent a powerful alternative to FISH for gene mapping because of its ability to generate multiple independent signals within the same gene segment. We investigated the specificity of signals produced by a modified PRINS protocol combining a centromeric probe for the X‐chromosome with specific primers for 3′‐ and 5′‐terminal regions of the dystrophin gene. In approximately 70% of nuclei from male and female subjects, we detected one or two large signals (X‐chromosome centromere) and two or four smaller signals (the two regions of the dystrophin gene). Specific hybridization of the oligonucleotides on Xp was demonstrated by localization of the smaller (dystrophin) and larger (X‐centromere) signals on the same chromosome. Simultaneous hybridization with a centromeric probe and gene‐specific oligonucleotides allowed localization of PRINS signals, and assessment of the specificity of the primers used for hybridization. This approach could facilitate identification of female carriers of small intragenic deletions in the dystrophin gene. © 2001 Wiley‐Liss, Inc.     

Åland Island eye disease (Forsius-Eriksson ocular albinism) and an Xp21 deletion in a patient with duchenne muscular dystrophy,glycerol kinase deficiency,and congenital adrenal hypoplasia

Glycerol kinase deficiency (GKD) has been described in isolation and in complex phenotypes including either congenital adrenal hypoplasia, Duchenne muscular dystrophy, or both. Cytogenetic and molecular studies have localized these defects to a deletion in volving the X chromosome at band Xp21, consistent with its X-linked recessive pattern of inheritance. Other clinical findings in the complex glycerol kinase deficiency (CGKD) patients are mental retardation, short stature, and hypogonadotropic hypogonadisem. We report on a 6-year-old boy who, in addition to the CGKD phenotype described above, had ocular hypopigmentation consistent with Forsius-Eriksson ocular albinism, also known as type 2 ocular albinism or Åland Island eye disease. Cytogenetic analysis shows an interstitial deletion in the short arm of the X-chromosome at Xp21.     

A pregnancy following PGD for X-linked dominant [correction of X-linked autosomal dominant] incontinentia pigmenti (Bloch-Sulzberger syndrome): case report

Incontinentia Pigmenti (Bloch-Sulzberger syndrome) is a rare multisystem, ectodermal disorder associated with dermatological, dental and ocular features, and in <10% of cases, severe neurological deficit. Pedigree review suggests X-linked dominance with lethality in affected males. Presentation in female carriers is variable. Following genetic counselling, a mildly affected female carrier diagnosed in infancy with a de novo mutation was referred for preimplantation sexing, unusually selecting for male gender, with an acceptance of either normality or early miscarriage in an affected male. Following standard in-vitro fertilization and embryo biopsy, fluorescence in situ hybridization (FISH) unambiguously identified two male and two female embryos. A single 8-cell, grade 4 male embryo was replaced. A positive pregnancy test was reported 2 weeks after embryo transfer, although ultrasonography failed to demonstrate a viable pregnancy. Post abortive fetal tissue karyotyping diagnosed a male fetus with trisomy 16. This is an unusual report of preimplantation genetic diagnosis (PGD) being used for selection of males in an X-linked autosomal dominant disorder and demonstrates the value of PGD where amniocentesis or chorion villus sampling followed by abortion is not acceptable to the patient. This case also demonstrates the importance of follow-up prenatal diagnosis.     

Clinical implications of fluorescence in situ hybridization analysis in 13 chronic myeloid leukemia cases: Ph-negative and variant Ph-positive.

Thirteen chronic myeloid leukemia (CML) patients, 10 with variant Philadelphia (Ph) translocations and 3 Ph negative cases, were analyzed by fluorescence in situ hybridization (FISH) with the use of BCR and ABL cosmid probes and a chromosome 22 painting probe. In the variant Ph translocations, the BCR-ABL fusion gene was located on the Ph chromosome; in 1 CML Ph-negative patient, the BCR-ABL fusion gene was located on the Ph chromosome; and, in 2 patients, it was located on chromosome 9. The chromosome 22 painting probe was detected on the third-party chromosome of the variant translocation, and in none of the variant translocations was there any detectable signal on chromosome 9. In CML patients with clonal evolution of a simple Ph, a signal of the chromosome 22 painting probe was detected on the der(9) of the Ph translocation. It was concluded that the variant Ph translocations evolved simultaneously in a three-way rearrangement. The clinical parameters of the 13 patients were similar to those of a large group of CML patients with a simple Ph translocation. It is suggested that, to determine the prognosis of CML patients with a complex karyotype, FISH analysis with a chromosome 22 painting probe be performed.     

Fabry disease in a large Nova Scotia kindred: carrier detection using leucocyte alpha-galactosidase activity and an NcoI polymorphism detected by an alpha-galactosidase cDNA clone.

Fabry disease is an X linked recessive disorder of glycosphingolipid metabolism resulting from a deficiency of the lysosomal hydrolase alpha-galactosidase (alpha-gal). Measurement of the enzyme activity, however, is not an accurate method for identification of female carriers among at risk relatives of affected males. The alpha-gal cDNA and gene have been cloned previously and found to provide useful probes for the molecular analysis of affected families but these clones have not been available to us. Thus, to analyse Fabry disease in Nova Scotia, especially within a large kindred known to contain 30 affected males and 50 possible carrier females, we isolated an independent cDNA for alpha-gal. Using this clone as a probe, the mutation in the Nova Scotia kindred was shown not to be a major DNA alteration, but was found to be linked to the rarer allele (frequency 0.20) of the polymorphic NcoI site located 3' to the gene. Affected males from two Nova Scotia families who cannot be associated with the kindred by history were also found to have the rarer NcoI allele, which suggests they are, in fact, part of the kindred. The coupling of the mutation to an infrequent marker also helped carrier identification in the kindred where all of 17 obligate carriers examined, including six who were not identified as carriers by enzyme assays, were found to be heterozygous for the RFLP. Thus, DNA analysis can be used for presymptomatic and prenatal diagnosis in most portions of the Nova Scotia kindred affected with Fabry disease.