Alu-Mediated Insertions in the DMD Gene: A Difficult Puzzle to Interpret Clinically.

Disrupting variants in the DMD gene are associated with Duchenne or Becker muscular dystrophy (DMD/BMD) or with hyperCKemia, all of which present very different degrees of clinical severity. The clinical phenotypes of these disorders could not be distinguished in infancy or early childhood. Accurate phenotype prediction based on DNA variants may therefore be required in addition to invasive tests, such as muscle biopsy. Transposon insertion is one of the rarest mutation types. Depending on their position and characteristics, transposon insertions may affect the quality and/or quantity of dystrophin mRNA, leading to unpredictable alterations in gene products. Here, we report the case of a three-year-old boy showing initial skeletal muscle involvement in whom we characterized a transposon insertion (Alu sequence) in exon 15 of the DMD gene. In similar cases, the generation of a null allele is predicted, resulting in a DMD phenotype. However, mRNA analysis of muscle biopsy tissue revealed skipping of exon 15, which restored the reading frame, thus predicting a milder phenotype. This case is similar to very few others already described in the literature. This case further enriches our knowledge of the mechanisms perturbing splicing and causing exon skipping in DMD, helping to properly guide clinical diagnosis.

Bi-Allelic DES Gene Variants Causing Autosomal Recessive Myofibrillar Myopathies Affecting Both Skeletal Muscles and Cardiac Function.

Mutations in the human desmin gene (DES) may cause both autosomal dominant and recessive cardiomyopathies leading to heart failure, arrhythmias and atrio-ventricular blocks, or progressive myopathies. Cardiac conduction disorders, arrhythmias and cardiomyopathies usually associated with progressive myopathy are the main manifestations of autosomal dominant desminopathies, due to mono-allelic pathogenic variants. The recessive forms, due to bi-allelic variants, are very rare and exhibit variable phenotypes in which premature sudden cardiac death could also occur in the first or second decade of life. We describe a further case of autosomal recessive desminopathy in an Italian boy born of consanguineous parents, who developed progressive myopathy at age 12, and dilated cardiomyopathy four years later and died of intractable heart failure at age 17. Next Generation Sequencing (NGS) analysis identified the homozygous loss-of-function variant c.634C>T; p.Arg212*, which was likely inherited from both parents. Furthermore, we performed a comparison of clinical and genetic results observed in our patient with those of cases so far reported in the literature.

Study of expression of genes potentially responsible for reduced fitness in patients with myotonic dystrophy type 1 and identification of new biomarkers of testicular function.

Myotonic dystrophy type 1 (DM1) is a multisystemic disorder caused by trinucleotide CTG expansion in DMPK gene, often affecting the neighboring genes. Endocrine system is involved, resulting in hypogonadism and reproductive abnormalities, but molecular mechanisms underlying the reduced fertility observed in DM1 are very complex and partially unknown. To better characterize these mechanisms, an analysis of sperm parameters and anti-Müllerian hormone (AMH) values was performed in 20 DM1 patients. About 50% of them showed hypoposia and azoospermia; the remaining, despite an adequate volume of ejaculate, had oligo-astheno-teratozoospermia. Interestingly, the lowest AMH levels better correlated with the main sperm alterations. The pattern of expression of DMPK, SIX5, and RSPH6A genes, evaluated by quantitative reverse transcription polymerase chain reaction, showed a substantial reduction of the expression in both peripheral blood and in seminal plasma of patients, compared to controls. An impairment of testis-specific RSPH6A protein expression and localization was observed in sperm protein extracts by WB analysis and in isolated spermatozoa by immunofluorescence. These results support the hypothesis that CTG expansion also affects the expression of neighboring genes and contributes to gonad defects observed in DM1, suggesting the possibility of using them as markers for normal fertility in humans.

Cross-sectional serum metabolomic study of multiple forms of muscular dystrophy.

Muscular dystrophies are characterized by a progressive loss of muscle tissue and/or muscle function. While metabolic alterations have been described in patients'-derived muscle biopsies, non-invasive readouts able to describe these alterations are needed in order to objectively monitor muscle condition and response to treatment targeting metabolic abnormalities. We used a metabolomic approach to study metabolites concentration in serum of patients affected by multiple forms of muscular dystrophy such as Duchenne and Becker muscular dystrophies, limb-girdle muscular dystrophies type 2A and 2B, myotonic dystrophy type 1 and facioscapulohumeral muscular dystrophy. We show that 15 metabolites involved in energy production, amino acid metabolism, testosterone metabolism and response to treatment with glucocorticoids were differentially expressed between healthy controls and Duchenne patients. Five metabolites were also able to discriminate other forms of muscular dystrophy. In particular, creatinine and the creatine/creatinine ratio were significantly associated with Duchenne patients performance as assessed by the 6-minute walk test and north star ambulatory assessment. The obtained results provide evidence that metabolomics analysis of serum samples can provide useful information regarding muscle condition and response to treatment, such as to glucocorticoids treatment.

Skewed X-chromosome inactivation plays a crucial role in the onset of symptoms in carriers of Becker muscular dystrophy.

BACKGROUND: Becker muscular dystrophy (BMD) is an X-linked recessive disorder affecting approximately 1: 18.000 male births. Female carriers are usually asymptomatic, although 2.5-18% may present muscle or heart symptoms. In the present study, the role of the X chromosome inactivation (XCI) on the onset of symptoms in BMD carriers was analysed and compared with the pattern observed in Duchenne muscular dystrophy (DMD) carriers. METHODS: XCI was determined on the lymphocytes of 36 BMD carriers (both symptomatic and not symptomatic) from 11 families requiring genetic advice at the Cardiomyology and Medical Genetics of the Second University of Naples, using the AR methylation-based assay. Carriers were subdivided into two groups, according to age above or below 50 years. Seven females from the same families known as noncarriers were used as controls. A Student's t-test for nonpaired data was performed to evaluate the differences observed in the XCI values between asymptomatic and symptomatic carriers, and carriers aged above or below 50 years. A Pearson correlation test was used to evaluate the inheritance of the XCI pattern in 19 mother-daughter pairs. RESULTS: The results showed that symptomatic BMD carriers had a skewed XCI with a preferential inactivation of the X chromosome carrying the normal allele, whereas the asymptomatic carriers and controls showed a random XCI. No concordance concerning the XCI pattern was observed between mothers and related daughters. CONCLUSIONS: The data obtained in the present study suggest that the onset of symptoms in BMD carriers is related to a skewed XCI, as observed in DMD carriers. Furthermore, they showed no concordance in the XCI pattern inheritance.

Determining the role of skewed X-chromosome inactivation in developing muscle symptoms in carriers of Duchenne muscular dystrophy.

Duchenne and Becker dystrophinopathies (DMD and BMD) are X-linked recessive disorders caused by mutations in the dystrophin gene that lead to absent or reduced expression of dystrophin in both skeletal and heart muscles. DMD/BMD female carriers are usually asymptomatic, although about 8 % may exhibit muscle or cardiac symptoms. Several mechanisms leading to a reduced dystrophin have been hypothesized to explain the clinical manifestations and, in particular, the role of the skewed XCI is questioned. In this review, the mechanism of XCI and its involvement in the phenotype of BMD/DMD carriers with both a normal karyotype or with X;autosome translocations with breakpoints at Xp21 (locus of the DMD gene) will be analyzed. We have previously observed that DMD carriers with moderate/severe muscle involvement, exhibit a moderate or extremely skewed XCI, in particular if presenting with an early onset of symptoms, while DMD carriers with mild muscle involvement present a random XCI. Moreover, we found that among 87.1 % of the carriers with X;autosome translocations involving the locus Xp21 who developed signs and symptoms of dystrophinopathy such as proximal muscle weakness, difficulty to run, jump and climb stairs, 95.2 % had a skewed XCI pattern in lymphocytes. These data support the hypothesis that skewed XCI is involved in the onset of phenotype in DMD carriers, the X chromosome carrying the normal DMD gene being preferentially inactivated and leading to a moderate-severe muscle involvement.

A rare co-occurrence of phosphorylase kinase deficiency (GSD type IXd) and alpha-glycosidase deficiency (GSD Type II) in a 53-year-old man presenting with an atypical glycogen storage disease phenotype.

Glycogen Storage Disease (GSD) IXd, caused by PHKA1 gene mutations, is an X-linked rare disorder that can be asymptomatic or associated with exercise intolerance. GSD type II is an autosomal recessive disorder caused by mutations in the GAA gene that lead to severe cardiac and skeletal muscle myopathy. We report the first case of co-occurrence of type IXd and type II GSDs in a 53-year-old man with an atypical glycogen storage disease presentation consisting in myalgia in the lower limbs at both rest and after exercise and increased levels of transaminases from the age of 16. At the age of 43, the patient presented a steppage gait, inability to run and walk on his heels, hypotrophy of the pectoral and proximal muscles, reflexes not elicitable, and CK levels 3.6 times the upper reference limit. Next Generation Sequencing (NGS) identified one variant in the PHKA1 gene, c.1360A > G p.Ile454Val (exon 14) inherited by his mother, and two heterozygous variants in the GAA gene, c.784G > A (exon 4) and c.956-6T > C (exon 6). A review of GSD IXd cases reported to date in the literature is also provided.

Phenotypic Variability of Andersen-Tawil Syndrome Due to Allelic Mutation c.652C>T in the KCNJ2 Gene-A New Family Case Report.

Andersen-Tawil syndrome (ATS) is a multisystem channelopathy characterized by periodic paralysis, ventricular arrhythmias, prolonged QT interval, and facial dysmorphisms occurring in the first/second decade of life. High phenotypic variability and incomplete penetrance of the genes causing the disease make its diagnosis still a challenge. We describe a three-generation family with six living individuals affected by ATS. The proband is a 37-year-old woman presenting since age 16, with episodes of muscle weakness and cramps in the pre-menstrual period. The father, two brothers, one paternal uncle and one cousin also complained of cramps, muscle stiffness, and weakness. Despite normal serum potassium concentration, treatment with potassium, magnesium, and acetazolamide alleviated paralysis attacks suggesting a dyskalemic syndrome. Dysmorphic features were noted in the proband, only later. On the ECG, all but one had normal QT intervals. The affected males developed metabolic syndrome or obesity. The father had two myocardial infarctions and was implanted with an intracardiac cardioverter defibrillator (ICD). A genetic investigation by WES analysis detected the heterozygous pathogenic variant (NM_000891.2: c.652C>T, p. Arg218Trp) in the KCNJ2 gene related to ATS, confirmed by segregation studies in all affected members. Furthermore, we performed a review of cases with the same mutation in the literature, looking for similarities and divergences with our family case.

DAG1 haploinsufficiency is associated with sporadic and familial isolated or pauci-symptomatic hyperCKemia.

DAG1 encodes for dystroglycan, a key component of the dystrophin-glycoprotein complex (DGC) with a pivotal role in skeletal muscle function and maintenance. Biallelic loss-of-function DAG1 variants cause severe muscular dystrophy and muscle-eye-brain disease. A possible contribution of DAG1 deficiency to milder muscular phenotypes has been suggested. We investigated the genetic background of twelve subjects with persistent mild-to-severe hyperCKemia to dissect the role of DAG1 in this condition. Genetic testing was performed through exome sequencing (ES) or custom NGS panels including various genes involved in a spectrum of muscular disorders. Histopathological and Western blot analyses were performed on muscle biopsy samples obtained from three patients. We identified seven novel heterozygous truncating variants in DAG1 segregating with isolated or pauci-symptomatic hyperCKemia in all families. The variants were rare and predicted to lead to nonsense-mediated mRNA decay or the formation of a truncated transcript. In four cases, DAG1 variants were inherited from similarly affected parents. Histopathological analysis revealed a decreased expression of dystroglycan subunits and Western blot confirmed a significantly reduced expression of beta-dystroglycan in muscle samples. This study supports the pathogenic role of DAG1 haploinsufficiency in isolated or pauci-symptomatic hyperCKemia, with implications for clinical management and genetic counseling.

Xp21 contiguous gene deletion syndrome presenting as Duchenne muscular dystrophy and glycerol kinase deficiency associated with intellectual disability: case report and review literature.

The contiguous gene deletion syndromes (CGDS) are rare genomic disorders resulting from the deletion of large segments of DNA, manifested as the concurrence of apparently unrelated clinical features. A typical example of CGDS is Xp21 contiguous gene deletion syndrome that involves GK and its neigh-boring genes (usually DMD and NR0B1) and results in a complex phenotype, which is related to the size of deletion and involved genes. Development delay and intellectual disability are almost a constant feature of patients with CGDS. We report the case of a boy with Duchenne muscular dystrophy (DMD) and glycerol kinase deficiency (GKD) as part of the contiguous gene deletion syndrome Xp2.1, in association with intellectual disability (ID) in whom multiplex ligation-dependent probe amplification (MLPA) test first identified a hemizygous deletion involving the entire dystrophin gene. Subsequently, the array CGH study identified a maternally inherited hemizygous deletion of the Xp21.2-Xp21.1 region of approximately 3.7Mb that included both DMD and GK genes confirming the diagnosis of Xp21 CGDS. Moreover, we report a review of the cases published in the literature over the last 20 years, for which a better description of the genes involved in the syndrome was available. Intellectual disability does not appear as a constant feature of the syndrome, reiterating the concept that complex GKD syndrome results from small deletions that affect closely related but separate loci for DMD, GK and adrenal hypoplasia, rather than a single large deletion including all genes. This case highlights the importance of more in-depth genetic investigations in presence of apparently unrelated clinical findings, allowing an accurate diagnosis of contiguous gene deletion syndromes.

Spectrum of Genetic Variants in the Dystrophin Gene: A Single Centre Retrospective Analysis of 750 Duchenne and Becker Patients from Southern Italy.

Dystrophinopathies are X-linked recessive muscle disorders caused by mutations in the dystrophin (DMD) gene that include deletions, duplications, and point mutations. Correct diagnosis is important for providing adequate patient care and family planning, especially at this time when mutation-specific therapies are available. We report a large single-centre study on the spectrum of DMD gene variants observed in 750 patients analyzed for suspected Duchenne (DMD) or Becker (BMD) muscular dystrophy, over the past 30 years, at the Cardiomyology and Medical Genetics of the University of Campania. We found 534 (71.21%) large deletions, 73 (9.73%) large duplications, and 112 (14.93%) point mutations, of which 44 (5.9%) were small ins/del causing frame-shifts, 57 (7.6%) nonsense mutations, 8 (1.1%) splice site and 3 (0.4%) intronic mutations, and 31 (4.13%) non mutations. Moreover, we report the prevalence of the different types of mutations in patients with DMD and BMD according to their decade of birth, from 1930 to 2020, and correlate the data to the different techniques used over the years. In the most recent decades, we observed an apparent increase in the prevalence of point mutations, probably due to the use of Next-Generation Sequencing (NGS). In conclusion, in southern Italy, deletions are the most frequent variation observed in DMD and BMD patients followed by point mutations and duplications, as elsewhere in the world. NGS was useful to identify point mutations in cases of strong suspicion of DMD/BMD negative on deletions/duplications analyses. In the era of personalized medicine and availability of new causative therapies, a collective effort is necessary to enable DMD and BMD patients to have timely genetic diagnoses and avoid late implementation of standard of care and late initiation of appropriate treatment.

Digital health and Clinical Patient Management System (CPMS) platform utility for data sharing of neuromuscular patients: the Italian EURO-NMD experience.

BACKGROUND: The development of e-health technologies for teleconsultation and exchange of knowledge is one of the core purposes of European Reference Networks (ERNs), including the ERN EURO-NMD for rare neuromuscular diseases. Within ERNs, the Clinical Patient Management System (CPMS) is a web-based platform that seeks to boost active collaboration within and across the network, implementing data sharing. Through CPMS, it is possible to both discuss patient cases and to make patients' data available for registries and databases in a secure way. In this view, CPMS may be considered a sort of a temporary storage for patients' data and an effective tool for data sharing; it facilitates specialists' consultation since rare diseases (RDs) require multidisciplinary skills, specific, and outstanding clinical experience. Following European Union (EU) recommendation, and to promote the use of CPMS platform among EURO-NMD members, a twelve-month pilot project was set up to train the 15 Italian Health Care Providers (HCPs). In this paper, we report the structure, methods, and results of the teaching course, showing that tailored, ERN-oriented, training can significantly enhance the profitable use of the CPMS. RESULTS: Throughout the training course, 45 professionals learned how to use the many features of the CPMS, eventually opening 98 panels of discussion-amounting to 82% of the total panels included in the EURO-NMD. Since clinical, genetic, diagnostic, and therapeutic data of patients can be securely stored within the platform, we also highlight the importance of this platform as an effective tool to discuss and share clinical cases, in order to ease both case solving and data storing. CONCLUSIONS: In this paper, we discuss how similar course could help implementing the use of the platform, highlighting strengths and weaknesses of e-health for ERNs. The expected result is the creation of a "map" of neuromuscular patients across Europe that might be improved by a wider use of CPMS.

Improvement of survival in Duchenne Muscular Dystrophy: retrospective analysis of 835 patients.

Duchenne Muscular Dystrophy (DMD) is the most common muscle disease in children. Historically, DMD results in loss of ambulation between ages 7 and 13 years and death in the teens or 20s. In order to determine whether survival has improved over the decades and whether the impact of nocturnal ventilation combined with a better management of cardiac involvement has been able to modify the pattern of survival, we reviewed the notes of 835 DMD patients followed at the Naples Centre of Cardiomyology and Medical Genetics from 1961 to 2006. Patients were divided, by decade of birth, into 3 groups: 1) DMD born between 1961 and 1970; 2) DMD born between 1971 and 1980; 3) DMD born between 1981 and 1990; each group was in turn subdivided into 15 two-year classes, from 14 to 40 years of age. Age and causes of death, type of cardiac treatment and use of a mechanical ventilator were carefully analyzed.The percentage of survivors in the different decades was statistically compared by chi-square test and Kaplan-Meier survival curves analyses. A significant decade on decade improvement in survival rate was observed at both the age of 20, where it passed from 23.3% of patients in group 1 to 54% of patients in group 2 and to 59,8% in patients in group 3 (p < 0.001) and at the age of 25 where the survival rate passed from 13.5% of patients in group 1 to 31.6% of patients in group 2 and to 49.2% in patients in group 3 (p < 0.001).The causes of death were both cardiac and respiratory, with a prevalence of the respiratory ones till 1980s. The overall mean age for cardiac deaths was 19.6 years (range 13.4-27.5), with an increasing age in the last 15 years. The overall mean age for respiratory deaths was 17.7 years (range 11.6-27.5) in patients without a ventilator support while increased to 27.9 years (range 23-38.6) in patients who could benefit of mechanical ventilation.This report documents that DMD should be now considered an adulthood disease as well, and as a consequence more public health interventions are needed to support these patients and their families as they pass from childhood into adult age.

Autosomal dominant Ullrich congenital muscular dystrophy due to a de novo mutation in COL6A3 gene. A case report.

Mutations in the genes encoding collagen VI cause Bethlem myopathy (MIM 158810), Ullrich congenital muscular dystrophy (MIM 254090), and myosclerosis myopathy (MIM #255600). BM is a dominantly inherited disorder, characterised by proximal muscle weakness and joint contractures mainly involving the elbows, ankles, and fingers, which usually follows a relatively mild course. By contrast, UCMD is a severe muscular dystrophy characterized by early onset, rapidly progressive muscle wasting and weakness, proximal joint contractures and distal joint hyperlaxity. Rapid progression usually leads to early death due to respiratory failure. UCMD is usually inherited as an autosomal recessive trait though dominant de novo heterozygous variants have recently been reported. We describe a further patient with UCMD classical presentation who showed, at the NGS analysis, the de novo variant c.6210+1G > A in the intron 16 of the gene COL6A3, known in the literature as pathogenic (VCV0000949S6.5).

Genetic counseling in Pompe disease.

Pompe disease is caused by glycogen accumulation due to a deficiency of the lysosomal acid alpha-glucosidase enzyme by which it is degraded. It is a rare disease, accounting for 1:40.000 births. It is inherited as an autosomal recessive trait so that a couple presents a recurrent risk of 25% to have a child affected, at each pregnancy. The diagnosis could be achieved by biochemical and/or molecular testing. Carrier detection and prenatal diagnosis are available when the molecular defect is known.

Cardiac involvement in patients with spinal muscular atrophies.

The spinal muscular atrophies (SMAs) include a group of disorders characterized by progressive weakness of the lower motor neurons. Several types of SMAs have been described based on age onset of clinical features: Acute infantile (SMA type I), chronic infantile (SMA type II), chronic juvenile (SMA type III), and adult onset (SMA type IV) forms. The incidence is about 1:6,000 live births with a carrier frequency of 1:40 for the severe form and 1:80 for the juvenile form. The mortality and/or morbidity rates of SMAs are inversely correlated with the age at onset. SMAs are believed to only affect skeletal muscles; however, new data on SMA mice models suggest they may also impact the heart. Aim of the study was to retrospectively examine the cardiological records of 37 type molecularly confirmed II/III SMA patients, aged 6 to 65 years, in order to evaluate the onset and evolution of the cardiac involvement in these disorders. All patients had a standard ECG and a routine echocardiography. The parameters analysed were the following: Heart rate (HR), PQ interval, PQ segment, Cardiomyopathic Index (ratio QT/PQs), ventricular and supraventricular ectopic beats, pauses > or = 2,5 msec, ventricle diameters, wall and septum thickness, ejection fraction, fiber shortening. The results showed that HR and the other ECG parameters were within the normal limits except for the Cardiomyopathic Index that was higher than the normal values (2,6-4,2) in 2 patients. Left ventricular systolic function was within the normal limits in all patients. A dilation of the left ventricle without systolic dysfunction was observed in only 2 patients, aged respectively 65 and 63 years; however they were hypertensive and/or affected by coronary artery disease. Data here reported contribute to reassure patients and their clinicians that type II/III SMAs do not present heart dysfunction.

CLCN1 Molecular Characterization in 19 South-Italian Patients With Dominant and Recessive Type of Myotonia Congenita.

Myotonia congenita is a genetic disease characterized by impaired muscle relaxation after forceful contraction (myotonia). It is caused by mutations in the CLCN1 gene, encoding the voltage-gated chloride channel of skeletal muscle, ClC-1. According to the pattern of inheritance, two distinct clinical forms have been described, Thomsen disease, inherited as an autosomal dominant trait and Becker disease inherited as an autosomal recessive trait. We report genetic and clinical data concerning 19 patients-13 familial and six isolated cases-all but one originating from the Campania Region, in southern Italy. Twelve patients (63.2%) present Becker type myotonia and 7 (36.8%) Thomsen type. Sex ratio M:F in Becker type is 6:6, while in Thomsen myotonia 4:3. The age of onset of the disease ranged from 2 to 15 years in Becker patients, and from 4 to 20 years in Thomsen. Overall 18 mutations were identified, 10 located in the coding part of the gene (exons 1, 3, 4, 5, 7, 8, 13, 15, 21, 22), and four in the intron part (introns 1, 2, 10, 18). All the exon mutations but two were missense mutations. Some of them, such as c.2551 G > A, c.817G > A and c.86A > C recurred more frequently. About 70% of mutations was inherited with an autosomal recessive pattern, two (c.86A and c.817G>A) with both mechanisms. Three novel mutations were identified, never described in the literature: p.Gly276Ser, p.Phe486Ser, and p.Gln812*, associated with Becker phenotype. Furthermore, we identified three CLCN1 mutations-c.86A>C + c.2551G > A, c.313C > T + c.501C > G and 899G > A + c.2284+5C > T, two of them inherited in cis on the same allele, in three unrelated families. The concomitant occurrence of both clinical pictures-Thomsen and Becker-was observed in one family. Intra-familial phenotypic variability was observed in two families, one with Becker phenotype, and one with Thomsen disease. In the latter an incomplete penetrance was hypothesized.

X-Linked Emery-Dreifuss Muscular Dystrophy: Study Of X-Chromosome Inactivation and Its Relation with Clinical Phenotypes in Female Carriers.

X-linked Emery-Dreifuss muscular dystrophy (EDMD1) affects approximately 1:100,000 male births. Female carriers are usually asymptomatic but, in some cases, they may present clinical symptoms after age 50 at cardiac level, especially in the form of conduction tissue anomalies. The aim of this study was to evaluate the relation between heart involvement in symptomatic EDMD1 carriers and the X-chromosome inactivation (XCI) pattern. The XCI pattern was determined on the lymphocytes of 30 symptomatic and asymptomatic EDMD1 female carriers-25 familial and 5 sporadic cases-seeking genetic advice using the androgen receptor (AR) methylation-based assay. Carriers were subdivided according to whether they were above or below 50 years of age. A variance analysis was performed to compare the XCI pattern between symptomatic and asymptomatic carriers. The results show that 20% of EDMD1 carriers had cardiac symptoms, and that 50% of these were ≥50 years of age. The XCI pattern was similar in both symptomatic and asymptomatic carriers. Conclusions: Arrhythmias in EDMD1 carriers poorly correlate on lymphocytes to a skewed XCI, probably due to (a) the different embryological origin of cardiac conduction tissue compared to lymphocytes or (b) the preferential loss of atrial cells replaced by fibrous tissue.

Interferon-gamma (INF-gamma), IL4 expression levels and Leishmania DNA load as prognostic markers for monitoring response to treatment of leishmaniotic dogs with miltefosine and allopurinol.

In this study, we searched for a connection between Leishmania load and cytokine expression levels in the tissues of Leishmaniainfantum naturally infected dogs and the efficacy of treatment with miltefosine and allopurinol. To this purpose, we exploited a real-time PCR system to detect Leishmania load and the expression levels of IFN-gamma and IL-4 mRNAs at the time of diagnosis and during the follow up post-treatment. In particular, we measured the amount of parasites in blood and lymph node samples, while the expression levels of IFN-gamma and IL-4 cytokines were assessed in the blood of the animals. We employed different targeted real-time PCR assays on 20 naturally infected dogs with clinical signs. Three healthy dogs living in a non-endemic area were selected as negative controls. The overall results obtained demonstrate that the simultaneous evaluation of parasites and cytokine levels in different kinds of tissue might represent a reliable tool to evaluate the immune response, the efficacy of the therapy and to predict the relapses during the treatment.

Urine sampling for real-time polymerase chain reaction based diagnosis of canine leishmaniasis.

A real-time polymerase chain reaction (PCR) assay was used for quantifying Leishmania infantum DNA in urine samples from naturally infected dogs. Forty-one infected dogs were divided into 3 groups: 22 dogs showing only cutaneous signs (group 1), 12 dogs showing hematuria (group 2), and 7 dogs affected by severe nephropathy (group 3). Groups 2 and 3 dogs showed altered laboratory parameters related to an impairment of renal function. The real-time PCR analysis showed higher levels of Leishmania DNA in the lymph node aspirates from all groups of infected dogs versus those measured in their blood or urine. Interestingly, urine samples from dogs belonging to groups 2 and 3 contained a higher Leishmania DNA load than that detected in their blood. This finding suggests that a real-time PCR analysis of urine from infected dogs could be a useful and noninvasive tool for monitoring the severity of leishmaniasis.