Facioscapulohumeral muscular dystrophy (FSHD) molecular diagnosis: from traditional technology to the NGS era.

Facioscapulohumeral muscular dystrophy (FSHD) is a genetic neuromuscular disorder which mainly affects the muscles of the face, shoulder, and upper arms. FSHD is generally associated with the contraction of D4Z4 macrosatellite repeats on 4q35 chromosome or mutations in SMCHD1, which are responsible of the toxic expression of DUX4 in muscle tissue. Despite the recent application of NGS techniques in the clinical practice, the molecular diagnosis of FSHD is still performed with dated techniques such as Southern blotting. The diagnosis of FSHD requires therefore specific skills on both modern and less modern analytical protocols. Considering that clinical and molecular diagnosis of FSHD is challenging, it is not surprising that only few laboratories offer a comprehensive characterization of FSHD, which requires the education of professionals on traditional techniques even in the era of NGS. In conclusion, the study of FSHD provides an excellent example of using classical and modern molecular technologies which are equally necessary for the analysis of DNA repetitive traits associated with specific disorders.

Migrainous Infarction in a Patient With Sporadic Hemiplegic Migraine and Cystic Fibrosis: A 99mTc-HMPAO Brain SPECT Study.

Genetic mutations of sporadic hemiplegic migraine (SHM) are mostly unknown. SHM pathophysiology relies on cortical spreading depression (CSD), which might be responsible for ischemic brain infarction. Cystic fibrosis (CF) is caused by a monogenic mutation of the chlorine transmembrane conductance regulator (CFTR), possibly altering brain excitability. We describe the case of a patient with CF, who had a migrainous stroke during an SHM attack. A 32-year-old Caucasian male was diagnosed with CF, with heterozygotic delta F508/unknown CFTR mutation. The patient experiences bouts of coughing sometimes triggering SHM attacks with visual phosphenes, aphasia, right-sided paresthesia, and hemiparesis. He had a 48-hour hemiparesis triggered by a bout of coughing with hemoptysis, loss of consciousness, and severe hypoxia-hypercapnia. MRI demonstrated transient diffusion hyperintensity in the left frontal-parietal-occipital regions resulting in a permanent infarction in the primary motor area. Later, a brain perfusion SPECT showed persistent diffuse hypoperfusion in the territories involved in diffusion-weighted imaging alteration. Migrainous infarction, depending on the co-occurrence of 2 strictly related phenomena, CSD and hypoxia, appears to be the most plausible explanation. Brain SPECT hypoperfusion suggests a more extensive permanent neuronal loss in territories affected by aura. CF may be then a risk factor for hemiplegic migraine and stroke since bouts of coughing can facilitate brain hypoxia, triggering auras.

Identification of Duchenne/Becker muscular dystrophy mosaic carriers through a combined DNA/RNA analysis.

OBJECTIVE: The Duchenne/Becker muscular dystrophy (DMD) carrier screening includes the evaluation of mutations in DMD gene, and the most widely used analysis is the multiplex ligation-dependent probe amplification (MLPA) for the DMD deletions/duplications detection. The high frequency of de novo mutations permits to estimate a risk up to 20% of mosaicisms for mothers of sporadic DMD children. The purpose of this study is to evaluate alternative analytical strategy for the detection of mosaics carrier women, in order to improve the recurrence risk estimation. METHOD: Different DNA and RNA analyses were conducted on samples from a woman that conceived a DMD fetus without previous family history of dystrophynopathy. RESULTS: Standard MLPA analysis failed to identify mosaicism, even if MLPA doses suggested it. Electrophoresis and direct sequencing conducted on RNA permitted to detect two different amplicons of cDNAs, demonstrating the presence of somatic mosaicism. Subsequent detection of a second affected fetus confirmed the mosaic status on the mother. CONCLUSION: The implementation of RNA analysis in diagnostic algorithm can increase the sensitivity of carrier test for mothers of sporadic affected patients, permitting detection of mosaic status. A revision of analytical guidelines is needed in order to improve the recurrence risk estimation and support prenatal genetic counseling.

Interpreting Mixture Profiles: Comparison between Precision ID GlobalFiler™ NGS STR Panel v2 and Traditional Methods.

Forensic investigation for the identification of offenders, recognition of human remains, and verification of family relationships requires the analysis of particular types of highly informative DNA markers, which have high discriminatory power and are efficient for typing degraded samples. These markers, called STRs (Short Tandem Repeats), can be amplified by multiplex-PCR (Polymerase Chain Reaction) allowing attainment of a unique profile through which it is possible to distinguish one individual from another with a high statistical significance. The rapid and progressive evolution of analytical techniques and the advent of Next-Generation Sequencing (NGS) have completely revolutionized the DNA sequencing approach. This technology, widely used today in the diagnostic field, has the advantage of being able to process several samples in parallel, producing a huge volume of data in a short time. At this time, although default parameters of interpretation software are available, there is no general agreement on the interpretation rules of forensic data produced via NGS technology. Here we report a pilot study aimed for a comparison between NGS (Precision ID GlobalFiler™ NGS STR Panel v2, Thermo Fisher Scientific, Waltham, MA, USA) and traditional methods in their ability to identify major and minor contributors in DNA mixtures from saliva and urine samples. A quantity of six mixed samples were prepared for both saliva and urine samples from donors. A total of 12 mixtures were obtained in the ratios of 1:2; 1:4; 1:6; 1:8; 1:10; and 1:20 between minor and major contributors. Although the number of analyzed mixtures is limited, our results confirm that NGS technology offers a huge range of additional information on samples, but cannot ensure a higher sensitivity in respect to traditional methods. Finally, the Precision ID GlobalFiler™ NGS STR Panel v2 is a powerful method for kinship analyses and typing reference samples, but its use in biological evidence should be carefully considered on the basis of the characteristics of the evidence.

NGS Analysis for Molecular Diagnosis of Retinitis Pigmentosa (RP): Detection of a Novel Variant in PRPH2 Gene.

This work describes the application of NGS for molecular diagnosis of RP in a family with a history of severe hypovision. In particular, the proband received a clinical diagnosis of RP on the basis of medical, instrumental examinations and his family history. The proband was subjected to NGS, utilizing a customized panel including 24 genes associated with RP and other retinal dystrophies. The NGS analysis revealed a novel missense variant (c.668T > A, I223N) in PRPH2 gene, which was investigated by segregation and bioinformatic analysis. The variant is located in the D2 loop domain of PRPH2, which is critical for protein activity. Bioinformatic analysis described the c.668T > A as a likely pathogenic variant. Moreover, a 3D model prediction was performed to better characterize the impact of the variant on the protein, reporting a disruption of the α-helical structures. As a result, the variant protein showed a substantially different conformation with respect to the wild-type PRPH2. The identified variant may therefore affect the oligomerization ability of the D2 loop and, ultimately, hamper PRPH2 proper functioning and localization. In conclusion, PRPH2_c.668T > A provided a molecular explanation of RP symptomatology, highlighting the clinical utility of NGS panels to facilitate genotype-phenotype correlations.

Limb-Girdle Muscular Dystrophies (LGMDs): The Clinical Application of NGS Analysis, a Family Case Report.

The diagnosis of LGMD2A (calpainopathy) can be challenging due to genetic heterogeneity and to high similarity with other LGMDs or neuromuscular disorders. In this setting, NGS panels are highly recommended to perform differential diagnosis, identify new causative mutations and enable genotype-phenotype correlations. In this manuscript, the case of a patient affected by LGMD2A is reported, for which the application of a defined custom designed NGS panel allowed to confirm the diagnosis of calpainopathy linked with two heterozygous variants in CAPN3, namely c.550delA and c.1813G>C. The first variant has been extensively described in relation to calpainopathy. The second variant c.1813G>C, instead, is novel and has been predicted to be probably damaging. In addition, NGS analysis on the proband revealed a heterozygous variant (c.550C>T) in the LMNA gene, which is associated with dilated cardiomyopathy. The variant is novel and has been predicted to be deleterious by subsequent bioinformatic analysis. Successively, segregation analysis was performed on family members. Interestingly, none of them showed neuromuscular symptoms but the mother was diagnosed with bradycardia and syncopal episodes and showed a positive family history for cardiomyopathy. The segregation analysis reported that the proband inherited the c.1813G>C (CAPN3) from the father who was a healthy carrier. The mother was positive for c.550delA (CAPN3) and c.550C>T (LMNA), suggesting thereby a possible genetic explanation for her cardiovascular problems. Segregation analysis, therefore, confirmed the inheritance pattern of the variants carried by the proband and highlighted a familiarity for cardiomyopathy which should not be neglected. The NGS analysis was further performed on the partner of the proband, to estimate the reproductive risk of the couple. The partner was negative to NGS screening, suggesting thereby a low risk to have an affected child with calpainopathy and 50% probability to inherit the LMNA variant. This case report showed the clinical utility of the NGS panel in providing accurate LGMD2A diagnosis and identifying complex phenotypes originating from mutations in multiple genes. However, NGS results should always be accomplished by a dedicated genetic counseling, not only to evaluate the recurrence and reproductive risks but also to uncover unexpected findings which can be clinically significant.

Erratum to "Mitochondrial Serine Protease HTRA2 p.G399S in a Female with Di George Syndrome and Parkinson's Disease".

[This corrects the article DOI: 10.1155/2018/5651435.].

Mitochondrial Serine Protease HTRA2 p.G399S in a Female with Di George Syndrome and Parkinson's Disease.

Deletion at 22q11.2 responsible for Di George syndrome (DGs) is a risk factor for early-onset Parkinson's disease (EOPD). To date, all patients reported with 22q11.2 deletions and parkinsonian features are negative for a family history of PD, and possible mutations in PD-related genes were not properly evaluated. The goal of this paper was to identify variants in PD genes that could contribute, together with 22q11.2 del, to the onset of parkinsonian features in patients affected by Di George syndrome. To this aim, sequencing analysis of 4800 genes including 17 PD-related genes was performed in a patient affected by DGs and EOPD. The analysis identified mutation p.Gly399Ser in OMI/HTRA2 (PARK13). To date, the mechanism that links DGs with parkinsonian features is poorly understood. The identification of a mutation in a PARK gene suggests that variants in PD-related genes, or in genes still not associated with PD, could contribute, together with deletion at 22q11.2, to the EOPD in patients affected by DGs. Further genetic analyses in a large number of patients are strongly required to understand this mechanism and to establish the pathogenetic role of p.Gly399Ser in OMI/HTRA2.