Carrier Frequency of Spinal Muscular Atrophy in a Large-scale Korean Population

Spinal muscular atrophy (SMA) is an autosomal recessive disease characterized by progressive proximal muscle weakness and atrophy. Given the recent introduction of gene therapies, knowledge of the SMA carrier frequency in various populations has become important for developing screening programs for this disease. In total, 1,581 anonymous DNA samples from an umbilical cord blood bank were tested for SMN1 and SMN2 gene copies using a multiplex ligation-dependent probe amplification assay. Twenty-nine of the 1,581 newborns [1.83%; 95% confidence interval (CI), 1.25–2.66%] were SMA carriers with one copy of SMN1, and no homozygous SMN1 deletion was detected. The carrier frequency in this population was estimated to be 1,834 per 100,000 (95% CI, 1,254–2,659) or 1 in 55 (95% CI, 1/79–1/38). Our data indicate that SMA carriers are not uncommon in the Korean population and may serve as a reference for designing a population screening program in Korea.

Targeted sequencing of the DMD locus: A comprehensive diagnostic tool for all mutations

Background & objectives: Duchenne muscular dystrophy (DMD) is an X-linked recessive disorder and is caused mainly by deletion, duplication and point mutations in the DMD gene. Diagnosis of DMD has been a challenge as the mutations in the DMD gene are heterogeneous and require more than one diagnostic strategy for the validation of the mutation. This study was planned to evaluate the targeted next-generation sequencing (NGS) as a single platform to detect all types of mutations in the DMD gene, thereby reducing the time and costs compared to conventional sequential testing and also provide precise genetic information for emerging gene therapies. Methods: The study included 20 unrelated families and 22 patients from an Indian population who were screened for DMD based on phenotypes such as scoliosis, toe walking and loss of ambulation. Peripheral blood DNA was isolated and subjected to multiplex ligation-dependent probe amplification (MLPA) and targeted NGS of the DMD gene to identify the nature of the mutation. Results: In the study patients, 77 per cent of large deletion mutations and 23 per cent single-nucleotide variations (SNVs) were identified. Novel mutations were also identified along with reported deletions, point mutations and partial deletions within the exon of the DMD gene. Interpretation & conclusions: Our findings showed the importance of NGS in the routine diagnostic practice in the identification of DMD mutations over sequential testing. It may be used as a single-point diagnostic strategy irrespective of the mutation type, thereby reducing the turnaround time and cost for multiple diagnostic tests such as MLPA and Sanger sequencing. Though MLPA is a sensitive technique and is the first line of a diagnostic test, the targeted NGS of the DMD gene may have an advantage of having a single diagnostic test. A study on a larger number of patients is needed to highlight NGS as a single, comprehensive platform for the diagnosis of DMD.

Detección de mutaciones causantes de distrofia muscular de Duchenne/Becker: reacción en cadena de la polimerasa Multiplex vs. Amplificación múltiple dependiente de ligación por sondas / Detection of mutations causing Duchenne and Becker muscular dystrophies: multiplex polymerase chain reaction vs. Multiplex ligation dependent probe amplification

RESUMEN Las distrofias musculares de Duchenne/Becker son enfermedades raras que reciben poca atención en nuestro medio. El objetivo del presente estudio fue implementar la técnica de amplificación múltiple dependiente de ligación por sondas (MLPA) y demostrar que tiene ventajas sobre la técnica de reacción en cadena de la polimerasa multiplex (PCR-multiplex). Se analizaron muestras de 40 individuos con diagnóstico presuntivo de distrofia muscular de Duchenne/Becker, primero por PCR-multiplex y luego por MLPA. Con la PCR-multiplex se detectaron 15 individuos con deleciones causales y con la técnica MLPA se logró diagnosticar a 21 individuos, cuatro duplicaciones y 17 deleciones. En conclusión, la técnica MLPA logra detectar mutaciones de tipo deleción y duplicación de exones, consiguiendo un mayor número de diagnósticos moleculares por alteraciones en el gen DMD.

ABSTRACT Duchenne and Becker muscular dystrophies are rare diseases that receive limited attention in our field. The objective of this study was to implement the Multiplex Ligation-dependent Probe Amplification technique (MLPA) and to demonstrate that it has advantages over the Multiplex Polymerase Chain Reaction (Multiplex PCR) technique. Samples from 40 individuals with a presumptive diagnosis of Duchenne and Becker muscular dystrophies were analyzed: first by Multiplex PCR and then by MLPA. Fifteen individuals with causal deletions were detected with Multiplex PCR, while the MLPA technique was able to diagnose 21 individuals, four duplications, and 17 deletions. In conclusion, the MLPA technique can detect mutations of the exon deletion and duplication type, yielding a larger number of molecular diagnoses due to alterations in the DMD gene.

Daily Iron Requirements in Healthy Indian Children and Adolescents

Objective: This study aimed to define the estimated average requirement and therecommended dietary allowance of iron for Indian children and adolescents. Methods: TheEstimated average requirement was derived for children aged 1-17y, from the meanbioavailability-adjusted daily physiological iron requirement, which in turn was estimatedusing a factorial method. This consisted of mean daily iron losses from the body andadditional iron required for tissue growth and storage, while also defining the variance of eachfactor to derive the Recommended dietary allowance. Results: The estimated averagerequirement of iron for children ranged from 5.6 to 11.0 mg/d in children aged 1-9y. Foradolescents aged 10-17y, these ranged from 10.8 to 18.4 mg/d and 15.4 to 18.5 mg/d foradolescent boys and girls, respectively. Conclusion: New estimates of estimated averagerequirement for iron in Indian children are presented, and same may be used to inform ironsupplementation and food fortification policies.

Supplementary value of denaturing high performance liquid chromatography for routine prenatal diagnosis of spinal muscular atrophy by multiple ligation-dependent probe amplification / 中华医学遗传学杂志

Objective@#To explore the feasibility of high performance liquid chromatography (DHPLC) combined with multiple ligation-dependent probe amplification (MLPA) for the prenatal diagnosis of spinal muscular atrophy (SMA).@*Methods@#Three families who had given birth to children with SMA type I were subjected to prenatal diagnosis. Peripheral blood samples were collected from the three couples, and 10 mL amniotic fluid was taken for each fetus through amniocentesis at 16-24 gestational week. Following DNA extraction, maternal contamination was excluded by STR analysis. Copy numbers of the SMN genes were detected by denaturing high performance liquid chromatography (DHPLC). Relative copy number of SMN1, SMN2 and reference genes was detected with a MLPA P021 assay kit.@*Results@#The three couples were all found to harbor heterozygous deletion of exon 7 of the SMN1 gene by DHPLC. MLPA analysis also suggested that the three couples were all carriers of SMA mutations. The fetus of family 1 harbored homozygous deletion of exons 7 and 8 of the SMN1 gene, in addition with heterozygous deletion of exons 7 and 8 of the SMN2 gene, suggesting that the fetus had SMA. The fetus of family 2 also harbored homozygous deletion of exons 7 and 8 of the SMN1 gene, while the copy number of SMN2 gene was normal, suggesting that the fetus was a SMA patient too. The fetus of family 3 harbored heterozygous deletion of exons 7 and 8 of the SMN1 gene, in addition with heterozygous deletion of exons 7 and 8 of the SMN2 gene, suggesting that the fetus was a carrier.@*Conclusion@#DHPLC can effectively screen carriers of SMA mutations. Combined DHPLC and MLPA can provide accurate diagnosis for fetuses with a high risk for SMA.

Molecular Genetics of von Willebrand Disease in Korean Patients: Novel Variants and Limited Diagnostic Utility of Multiplex Ligation-Dependent Probe Amplification Analyses

BACKGROUND: von Willebrand disease (VWD), characterized by quantitative or qualitative defects of von Willebrand factor (VWF), is the most common inheritable bleeding disorder. Data regarding the genetic background of VWD in Korean patients is limited. To our knowledge, this is the first comprehensive molecular genetic investigation of Korean patients with VWD. METHODS: Twenty-two unrelated patients with VWD were recruited from August 2014 to December 2017 (age range 28 months–64 years; male:female ratio 1.2:1). Fifteen patients had type 1, six had type 2, and one had type 3 VWD. Blood samples were collected for coagulation analyses and molecular genetic analyses from each patient. Direct sequencing of all exons, flanking intronic sequences, and the promoter of VWF was performed. In patients without sequence variants, multiplex ligation-dependent probe amplification (MLPA) was performed to detect dosage variants. We adapted the American College of Medical Genetics and Genomics guidelines for variant interpretation and considered variants of uncertain significance, likely pathogenic variants, and pathogenic variants as putative disease-causing variants. RESULTS: VWF variants were identified in 15 patients (68%): 14 patients with a single heterozygous variant and one patient with two heterozygous variants. The variants consisted of 13 missense variants, one small insertion, and one splicing variant. Four variants were novel: p.S764Efs*16, p.C889R, p.C1130Y, and p.W2193C. MLPA analysis in seven patients without reportable variants revealed no dosage variants. CONCLUSIONS: This study revealed the spectrum of VWF variants, including novel ones, and limited diagnostic utility of MLPA analyses in Korean patients with VWD.

A novel mutation in XLRS1 gene in X-linked juvenile retinoschisis

X-linked juvenile retinoschisis (XLRS) is characterized by the progressive loss of visual acuity and vitreous hemorrhage. XLRS is caused by a mutation of retinoschisin 1 (RS1) gene at Xp22.13. In the current report, a 2-year-old Korean patient with XLRS was described. The germline deletion of exon 1 was identified in the RS1 gene. Considering X-linked inheritance pattern, validation of a carrier state of a patient's mother is important for the genetic counseling of other family members and for the future reproductive plan. To confirm the carrier state of his mother, the multiplex ligation-dependent probe amplification analysis was done using peripheral leukocytes and found the heterozygous deletion of exon 1 in his mother.

Molecular Analysis-Based Genetic Characterization of a Cohort of Patients with Duchenne and Becker Muscular Dystrophy in Eastern China / 中华医学杂志(英文版)

<p><b>Background</b>Duchenne muscular dystrophy (DMD) and Becker muscular dystrophy (BMD) are common X-linked recessive neuromuscular disorders caused by mutations in dystrophin gene. Multiplex polymerase chain reaction (multiplex PCR) and multiplex ligation-dependent probe amplification (MLPA) are the most common methods for detecting dystrophin gene mutations. This study aimed to contrast the two methods and discern the genetic characterization of patients with DMD/BMD in Eastern China.</p><p><b>Methods</b>We collected 121 probands, 64 mothers of probands, and 15 fetuses in our study. The dystrophin gene was detected by multiplex PCR primarily in 28 probands, and MLPA was used in multiplex PCR-negative cases subsequently. The dystrophin gene of the remaining 93 probands and 62 female potential carriers was tested by MLPA directly. In fetuses, multiplex PCR and MLPA were performed on 4 fetuses and 10 fetuses, respectively. In addition, sequencing was also performed in 4 probands with negative MLPA.</p><p><b>Results</b>We found that 61.98% of the subjects had genetic mutations including deletions (50.41%) and duplications (11.57%). There were 43.75% of mothers as carriers of the mutation. In 15 fetuses, 2 out of 7 male fetuses were found to be unhealthy and 2 out of 8 female fetuses were found to be carriers. Exons 3-26 and 45-52 have the maximum frequency in mutation regions. In the frequency of exons individually, exon 47 and exon 50 were the most common in deleted regions and exons 5, 6, and 7 were found most frequently in duplicated regions.</p><p><b>Conclusions</b>MLPA has better productivity and sensitivity than multiplex PCR. Prenatal diagnosis should be applied in DMD high-risk fetuses to reduce the disease incidence. Furthermore, it is the responsibility of physicians to inform female carriers the importance of prenatal diagnosis.</p>

Analysis of two Chinese Han families with Duchenne/Becker muscular dystrophy / 上海交通大学学报（医学版）

Objective: To deepen the understanding of Duchenne/Becker muscular dystrophy by investigating dystrophin (DMD) gene variants in 2 Chinese Han families with this disease. Methods: Retrospective analysis of the clinical characteristics of the probands in two families with Duchnne/ Becker muscular dystrophy and the results of multiplex ligation-dependent probe amplification (MLPA) for the probands and their relatives was performed. Results: Three probands were identified by significantly-elevated creatine kinase levels. Two probands in family one are fraternal twin brothers with the same deletions of exons 8-9, while their mother has no abnormality at this site. The proband in family two is the little brother in a pair of fraternal twins with duplication of exons 48-51, and his mother has heterozygous duplication of exons 48-51. Conclusion: ① The presence of the same DMD gene mutation in the fraternal twins suggests that the mother may be a gonad chimera with this mutation if her gene detection of peripheral blood is normal. The mother must undergo prenatal gene diagnosis to reduce the risk of Duchenne/Becker muscular dystrophy in her offsprings. ② The exons 48-51 duplication of DMD gene is pathogenic mutation.

Multiplex Ligation-dependent Probe Amplification Analysis Subsequent to Direct DNA Full Sequencing for Identifying ATP7B Mutations and Phenotype Correlations in Children with Wilson Disease

BACKGROUND: Mutations in ATP7B cause Wilson disease (WD). However, direct DNA full sequencing cannot detect all mutations in patients with WD. Multiplex ligation-dependent probe amplification (MLPA) analysis is reportedly useful in increasing the diagnostic yield in other genetic disorders with large deletions or insertions. The aim of this study was to evaluate whether the detection rate of ATP7B mutations can be increased by using MLPA. METHODS: We enrolled 114 children with WD from 104 unrelated families based on biochemical tests and direct DNA full sequencing. The patients with one or zero mutant allele were investigated using MLPA. We analyzed phenotypic correlations. RESULTS: Total allele frequency by full sequencing was 87.5%. Full sequencing revealed two mutant alleles in 80 of 104 unrelated children. One mutant allele was detected in 22 children, and no mutations were found in two children. Novel mutations including small deletions with frameshift mutations were identified by DNA sequencing. MLPA revealed no gross deletion or duplication in 24 children with one or zero mutant alleles. The number of detected mutations was not associated with hepatic manifestation, age of onset, Kayser-Fleischer ring, ceruloplasmin, and urinary Cu concentrations. CONCLUSION: MLPA showed a limited role to increase the mutation detection rate in children who do not receive a definite genetic diagnosis of WD through DNA full sequencing. This finding suggests that large deletions or duplications might be extremely rare in WD. Further development is needed to improve the genetic diagnosis of WD.

Rare α0-thalassemia deletions detected by MLPA in five unrelated Brazilian patients

Abstract Alpha-thalassemias are among the most common genetic diseases in the world. They are characterized by hypochromic and microcytic anemia and great clinical variability, ranging from a practically asymptomatic phenotype to severe anemia, which can lead to intrauterine or early neonatal death. Deletions affecting the α-globin genes, located on chromosome 16p13.3, are the main causes of α-thalassemia. Multiplex ligation-dependent probe amplification (MLPA) can be used to detect rearrangements that cause α-thalassemia, particularly large deletions involving the whole α cluster and/or deletions in the HS-40 region. Here, MLPA was used to investigate the molecular basis of α-thalassemia in five unrelated patients, three of whom had Hb H disease. In addition to the -α3.7 deletion identified in the patients with Hb H disease, four different α0 deletions removing 15 to 225 kb DNA segments were found: two of them remove both the α genes, one affects only the regulatory element (HS-40) region, and another one extends over the entire α cluster and the HS-40 region. These results illustrate the diversity of α-thalassemia deletions in the Brazilian population and highlight the importance of molecular investigation in cases that present with microcytosis and hypochromia without iron deficiency and normal or reduced Hb A2 levels..

Deletion and Duplication in the DMD Gene Detected by MLPA / 现代检验医学杂志

Objective To detect exon deletions/duplications in the DMD gene in Duchenne and Becker muscular dystrophy pedigrees using multiplex ligation-dependent probe amplification method,and explore the usefulness of multiplex ligation-dependent probe amplification analysis as a method for genetic diagnostics in patients with Duchenne and Becker muscular dystrophy.Methods Exon deletions/duplications in the DMD gene were analyzed by multiplex ligation-dependent probe amplification for two pedigrees with Duchenne muscular dystrophy and Becker muscular dystrophy.Patients and carriers were diagnosed by multiplex ligation-dependent probe amplification.Results The pedigree with Duchenne muscular dystrophy was caused by DelEx45-50 mutation,while the pedigree with Becker muscular dystrophy was caused by Dup Ex3-4 mutation.Patients and carriers were diagnosed by multiplex ligation-dependent probe amplification method.Conclusion Exon deletions/duplications in the DMD gene can be indicated by probe copies using multiplex ligation-dependent probe amplification method under standard operating procedure.Multiplex ligation-dependent probe amplification should be considered as a rapid and accurate clinical method for an initial mutation analysis of DMD gene with exon deletions/duplications.

The SLC22A5 genetic analysis in Chinese patients with systemic primary carnitine deficiency / 中华内分泌代谢杂志

Objective To investigate the clinical and biochemical metabolic features of 12 patients with systemic primary carnitine deficiency(CDSP) and to identify the SLC22A5 gene mutation types of the disease. Method The clinical and biochemical data were collected by retrospective analysis. DNA direct sequencing and multiplex ligation dependent probe amplification(MLPA)were applied for SLC22A5 gene analysis. Result Among 12 patients with CDSP, 3 cases had evident infection factors, 6 cases with convulsions, 5 cases manifested liver hypertrophy, 8 cases with hyperammonemia, and 9 cases showed myocardial damage. All CDSP patients were detected biallelic pathogenic mutation in SLC22A5 gene by direct sequencing. The gene types include IVS2+1G>T, c.3G>T(p.Met1Ile), c.760C>T(p.Arg254X), c.1400C>G(p.Ser467Cys), c.844dupc(p.Arg282fs), c.338G>A(p.Cys113Tyr), c.51C>G(p.Phe17Leu), c.659A>T(p.Glu220Val), and c.1365dupC(p.Thr456fs). c.659A>T(p.Glu220Val) and c.1365dupC(p.Thr456fs)are novel mutations. One female patient was maternal CDSP, her child had abnormal newborn screening. The allele frequency of c.760C>T(p.Arg254X) and c.1400C>G(p.Ser467Cys) were 37.5%(9/24)and 29.2%(7/24)respectively. The MLPA test results of all patients were negative. Conclusion The clinical manifestations are complex and various in patients with CDSP. Point and small InDel(insertions/deletions)mutation constitute the major alteration in SLC22A5 gene. c.1400C>G(p.Ser467Cys) might be another prevalence mutation type in Chinese CDSP patient.

Improving the Diagnosis of Children with 22q11.2 Deletion Syndrome: A Single-center Experience from Serbia.

Objective: The incidence of the 22q11.2 microdeletion among children who have at least two out of five major clinical criteria for 22q11.2 deletion syndrome. Design: Prospective study. Setting: University Children’s Hospital in Belgrade, Serbia between 2005 and 2014. Participants: 57 patients with clinical characteristics of 22q11.2 deletion syndrome. Methods: Standard G-banding cytogenetic analysis was performed in all children, and the 22q11.2 genomic region was examined using fluorescence in situ hybridization (FISH). For patients with no deletion detected by FISH, multiplex ligation-dependent probe amplification (MLPA) analysis was also done in order to detect cryptic deletions of this region and to analyze other genomic loci associated with phenotypes resembling the syndrome. A selected group of patients diagnosed to have 22q11.2 microdeletion by FISH underwent MLPA testing in order to characterize the size and position of deletion. Outcome Measure: The frequency of 22q11.2 microdeletion among children with at least two of the five major characteristics of 22q11.2 deletion syndrome (heart malformations, facial dysmorphism, T-cell immunodeficiency, palatal clefts and hypocalcemia/hypoparathyroidism) Results: Typical 22q11.2 microdeletion was detected in 42.1% of patients; heart malformation were identified in all of them, facial dysmorphism in 79.2%, immunological problems in 63.6%, hypocalcemia in 62.5% and cleft palate in 8.3%. Conclusions: A higher detection rate compared to one-feature criterion is obtained when at least two major features of 22q11.2 deletion syndrome are taking into consideration. The criteria applied in this study could be considered by centers in low-income countries.

Clinical and laboratory features and gene diagnosis of Menkes disease / 中华实用儿科临床杂志

Objective To explore the clinical and laboratory features,and gene diagnosis method of Menkes disease (MD).Methods The clinical and laboratory features and gene diagnosis method of 2 infants with MD were reviewed.Results (1) Clinical features:both infants mentioned in this article were male.Their clinical manifestations were both began at 3-4 months age,including peculiar kinky hair,pale skin,pudgy cheeks,inguinal hernia,vessel abnormality,epilepsy and mental retardation.(2) Laboratory features:the ceruloplasmin concentrations significantly reduced to be ＜ 20 mg/L and 47 mg/L,respectively.The magnetic resonance angiogram images of case 1 showed the abnormal tortuosity of his intracranial vessels.The magnetic resonance images of case 2 showed a rapid progress from normal to severe brain atrophy within half a year.(3) Gene diagnosis:the sequencing of ATP7A gene in case 1 showed a nonsense mutation of c.2110 C ＞ T.The pathogenicity of this mutation had not been reported previously at home and abroad.The sequencing of the gene panel without pathogenic mutation was detected in case 2.But the multiplex ligation-dependent probe amplification test showed a gross deletion of ATP7A gene containing 8-12 exons.This mutation had been documented as a pathogenic mutation of MD.Both mothers of 2 patients were heterozygous mutation carriers of normal phenotype.Conclusions MD is a multisystemic disease caused by ATP7A gene mutation resulting in copper metabolism disorder.MD is inherited as an X-linked recessive trait.MD is characterized by kinky hair,connective tissue abnormalities and progressive neurodegeneration.Clinical diagnosis can be made on the basis of clinical features,findings of blood biochemical examination,and radiological findings.Gene sequencing and multiplex ligation dependent probe amplification test are the main technique widely used for genetic diagnosis.

Application of MLPA for analysis of chromosomal unbalances / 国际检验医学杂志

Objective To valuate the applicability of multiplex ligation‐dependent probe amplication (MLPA ) for detection of chromosomal unbalance .Methods Aneuploid and subtelomeric MLPA technology were used to analyze 6 samples with chromosom‐al unbalances ,confirmed by karyotype analysis .Results All samples were identified to have abnormal signal of corresponding probes .The extent of unbalances contained subtelomeric region .Conclusion MLPA technology could have important role in diag‐nosis of chromosomal unbalance ,which could complement conventional karyotype analysis .

Association of FOXE1 gene polymorphism with idiopathic premature ovarian failure / 中华妇产科杂志

Objective To assess the influence of length of the alanine tract of forkhead box E1 (FOXE1) gene on genetic susceptibility to idiopathic premature ovarian failure (POF). Methods Totally 110 patients with idiopathic POF were recruited between February 2009 and December 2012 at the Affiliated Shenzhen City Maternity and Child Healthcare Hospital of Southern Medical University. Controls (n=110) were individuals with normal menstrual cycles, normal FSH concentrations. The polyalanine tract and flanking sequence of FOXE1 were screened using the multiplex ligation-dependent probe amplification (MLPA) technique and direct sequence technique. Results The most frequent of FOXE1 polyalanine stretch length was 14 residues in both groups. The length of FOXE1 polyalanine reported in this study varied from 12 to 16 alanines, and three variants of FOXE1-polyalanine length, containing 12, 14, or 16 alanine residues, and 5 different genotypes were identified. The most common genotypes were 14/14 homozygote, occurring with the frequency of 81.8% (90/110) in the POF group, while 96.4% (106/110) in control subjects, respectively. The incidence of 14/14 genotypes of FOXE1-polyalanine was significantly lower in patients with POF (χ2=119.730, P=0.001) in comparison to the controls. There were significantly higher frequencies of the 16/16 genotypes in cases with POF [10.0% (11/110) versus 0; χ2=3.403, P=0.001], as compared with the controls. The FOXE1 14 alanine allele was significantly less common in the POF patient group than the controls [84.5% (186/220) versus 98.2% (216/220); χ2=25.923, P=0.001]. The FOXE1 16 alanine allele was significantly more common in the POF patient group than the controls [12.7% (28/220) versus 1.8% (4/220); χ2=19.412, P=0.001]. Conclusions The polymorphism of the polyalanine tract of FOXE1 gene have a certain relevance for the genetic aetiology of idiopathic POF.

Novel Non-contiguous Duplications in the DMD Gene in Five Patients with Duchenne Muscular Dystrophy

BACKGROUND: Muscular dystrophy is an X-linked recessive disorder caused by mutations in the DMD gene. Muscular dystrophy is classified into 2 types; Duchenne muscular dystrophy (DMD), which has severe clinical symptoms, and Becker muscular dystrophy (BMD), which has much milder clinical symptoms. Phenotypic progression to either DMD or BMD can be predicted by analyzing mutations in DMD by using the reading frame rule. METHODS: Of 88 patients with mutations in DMD, which were detected using Multiplex Ligation-dependent Probe Amplification DMD test kit (MRC-Holland, The Netherlands), medical records of 5 patients with non-contiguous duplications were reviewed. These rare non-contiguous duplications in DMD were compared with those reported previously. RESULTS: We identified 3 novel non-contiguous duplications in DMD that included exons 2-7 and 45-51, exons 5-37 and 50-59, and exons 52-53 and 56-61. The 5 patients with these non-contiguous duplications showed the phenotypic features of DMD. Especially, duplication of exons 52-53 and 56-61 was observed in a family, i.e., 2 DMD-affected brothers and their carrier mother. CONCLUSIONS: Prediction of phenotypes associated with complex non-contiguous duplications by using the reading frame rule is difficult because the duplications affect the expression of DMD together. Because most patients with non-contiguous duplications showed the phenotypic features of DMD, the reading frame rule should be interpreted cautiously. This study provides important insights on the non-contiguous duplications in DMD for understanding genotype-phenotype correlations and for developing dystrophin for therapeutic purposes.

Clinical and Genetic Characterization of Female Dystrophinopathy

BACKGROUND AND PURPOSE: Duchenne and Becker muscular dystrophies are the most common X-linked recessive muscular dystrophies. Dystrophin gene mutations usually affect men, but reportedly 2.5-7.8% of women are affected and are classified as symptomatic carriers. The aim of this study was to clinically and genetically characterize symptomatic female dystrophinopathy carriers. METHODS: The clinical and genetic data of 11 female dystrophinopathy carriers among 285 patients who underwent multiplex ligation-dependent probe amplification (MLPA) analysis for the dystrophin gene were reviewed. Women with muscle weakness and/or dilated cardiomyopathy were classified as symptomatic carriers, while subjects with high serum creatine kinase (CK) levels and/or minor myopathic signs such as muscle cramps and myalgia were classified as asymptomatic. RESULTS: Twelve female carriers were identified, but 1 symptomatic carrier who also had Turner syndrome was excluded from the study. Of the 11 included female carriers, 4 were symptomatic and 7 were asymptomatic. The age at symptom onset in the symptomatic female carriers ranged from 15 to 31 years (mean, 30.6 years), and the age at diagnosis for asymptomatic carriers ranged from 4 to 38 years (mean, 24.5 years). Serum CK levels were markedly elevated (mean, 1,301 IU/mL) in three of the four (75%) symptomatic female carriers, and mildly elevated in three of the seven (42%) asymptomatic female carriers. Symptomatic female carriers typically presented with asymmetric bilateral leg weakness as the initial symptom, with aggravated symptoms after labor. CONCLUSIONS: Female dystrophinopathy is not uncommon, and it is an important factor with respect to males with dystrophinopathy who may be born to such patients. Screening with MLPA is useful because it can aid in early diagnosis and appropriate management.

Large Deletion in KCNQ1 Identified in a Family with Jervell and Lange-Nielsen Syndrome

Long QT syndrome (LQTS) is a genetically heterogeneous disorder associated with sequence variations in more than 10 genes; in some cases, it is caused by large deletions or duplications among the main, known LQTS-associated genes. Here, we describe a 14-month-old Korean boy with congenital hearing loss and prolonged QT interval whose condition was clinically diagnosed as Jervell and Lange-Nielsen syndrome (JLNS), a recessive form of LQTS. Genetic analyses using sequence analysis and multiplex ligation-dependent probe amplification (MLPA) assay revealed a large deletion spanning exons 7-10 as well as a frameshift mutation (c.1893dup; p.Arg632Glnfs*20). To our knowledge, this is the first report of a large deletion in KCNQ1 identified in JLNS patients. This case indicates that a method such as MLPA, which can identify large deletions or duplications needs to be considered in addition to sequence analysis to diagnose JLNS.