Generation of the First Human In Vitro Model for McArdle Disease Based on iPSC Technology.

McArdle disease is a rare autosomal recessive disorder caused by mutations in the PYGM gene. This gene encodes for the skeletal muscle isoform of glycogen phosphorylase (myophosphorylase), the first enzyme in glycogenolysis. Patients with this disorder are unable to obtain energy from their glycogen stored in skeletal muscle, prompting an exercise intolerance. Currently, there is no treatment for this disease, and the lack of suitable in vitro human models has prevented the search for therapies against it. In this article, we have established the first human iPSC-based model for McArdle disease. For the generation of this model, induced pluripotent stem cells (iPSCs) from a patient with McArdle disease (harbouring the homozygous mutation c.148C>T; p.R50* in the PYGM gene) were differentiated into myogenic cells able to contract spontaneously in the presence of motor neurons and generate calcium transients, a proof of their maturity and functionality. Additionally, an isogenic skeletal muscle model of McArdle disease was created. As a proof-of-concept, we have tested in this model the rescue of PYGM expression by two different read-through compounds (PTC124 and RTC13). The developed model will be very useful as a platform for testing drugs or compounds with potential pharmacological activity.

Noninvasive prenatal testing: How far can we reach detecting fetal copy number variations.

Non-invasive prenatal testing (NIPT) is currently the best screening test for fetal chromosome abnormalities with the highest sensitivity and specificity and can be done from 10 weeks gestation. We report a detection of 44.7 Mb duplication at 11p15.5-p11.2 by NIPT with a fetal fraction (FF) of only 3%. This chromosome abnormality was confirmed after amniocentesis by karyotyping and array comparative genomic hybridization (aCGH) on cultured fetal cells. Further parental investigation showed that the fetal chromosome abnormality was inherited from the mother who was a carrier of a balanced translocation 46,XX,t(11;X)(p11.2;q28). This case highlights the importance of expanded NIPT in the detection of fetal segmental aneuploidy. NIPT together with complementary studies can lead to the detection of parental chromosome rearrangement despite a low FF, which can impact the couple's reproductive plans. We also reviewed other cases with chromosome rearrangement, detected by NIPT, derived from a parental reciprocal translocation.

Generation of the iPSC line IISHDOi007-A from peripheral blood mononuclear cells from a patient with McArdle disease harbouring the mutation c.2392 T > C; p.Trp798Arg.

Peripheral blood mononuclear cells (PBMCs) from a McArdle patient carrying a homozygous mutation in the PYGM gene: c.2392 T > C; p.Trp798Arg were used for the generation of the human iPSC line, IISHDOi007-A. For the delivery of the reprogramming factors Oct3/4, Sox2, Klf4, and c-Myc, a non-integrative methodology that implies the use of Sendai virus has been applied.

Derivation of a human DOA iPSC line, IISHDOi006-A, with a mutation in the ACO2 gene: c.1999G>A; p.Glu667Lys.

Human iPSC line, IISHDOi006-A, was obtained from fibroblasts of a patient with Dominant Optic Atrophy (DOA) carrying a heterozygous mutation in the gene ACO2: c.1999G>A; p.Glu667Lys. Reprogramming factors Oct3/4, Sox2, Klf4, and c-Myc were delivered using a non-integrative methodology that involves the use of Sendai virus.

Establishment of a human iPSC line, IISHDOi004-A, from a patient with Usher syndrome associated with the mutation c.2276G>T; p.Cys759Phe in the USH2A gene.

A human iPSC line, IISHDOi004-A, from fibroblasts obtained from a patient with Usher syndrome, harboring a homozygous mutation in the USH2A gene (c.2276G>T; p.Cys759Phe) has been generated. Reprogramming factors Oct3/4, Sox2, Klf4, and c-Myc were delivered using Sendai virus.

Noninvasive Prenatal Testing: Comparison of Two Mappers and Influence in the Diagnostic Yield.

OBJECTIVE: The aim of this study was to determine if the use of different mappers for NIPT may vary the results considerably. METHODS: Peripheral blood was collected from 217 pregnant women, 58 pathological (34 pregnancies with trisomy 21, 18 with trisomy 18, and 6 with trisomy 13) and 159 euploid. MPS was performed following a manufacturer's modified protocol of semiconductor sequencing. Obtained reads were mapped with two different software programs: TMAP and HPG-Aligner, comparing the results. RESULTS: Using TMAP, 57 pathological samples were correctly detected (sensitivity 98.28%, specificity 93.08%): 33 samples as trisomy 21 (sensitivity 97.06%, specificity 99.45%), 16 as trisomy 18 (sensibility 88.89%, specificity 93.97%), and 6 as trisomy 13 (sensibility 100%, specificity 100%). 11 false positives, 1 false negative, and 2 samples incorrectly identified were obtained. Using HPG-Aligner, all the 58 pathological samples were correctly identified (sensibility 100%, specificity 96.86%): 34 as trisomy 21 (sensibility 100%, specificity 98.91%), 18 as trisomy 18 (sensibility 100%, specificity 98.99%), and 6 as trisomy 13 (sensibility 100%, specificity 99.53%). 5 false positives were obtained. CONCLUSION: Different mappers use slightly different algorithms, so the use of one mapper or another with the same batch file can provide different results.

Encefalopatía epiléptica del lactante: lo prioritario es un buen estudio genético / Infantile epileptic encephalopathy: a good genetic study should be a priority

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Generation of a human iPSC line, IISHDOi002-A, with a 46, XY/47, XYY mosaicism and belonging to an African mitochondrial haplogroup.

We have generated a human iPSC line, IISHDOi002-A, from commercial primary normal human dermal fibroblasts belonging to an African mitochondrial haplogroup (L3), and with a 46, XY/47, XYY mosaicism. For this purpose, reprogramming factors Oct3/4, Sox2, Klf4 and cMyc were delivered using a non-integrative methodology that involves the use of Sendai virus.

Establishment of a human DOA 'plus' iPSC line, IISHDOi003-A, with the mutation in the OPA1 gene: c.1635C>A; p.Ser545Arg.

We have generated a human iPSC line IISHDOi003-A from fibroblasts of a patient with a dominant optic atrophy 'plus' phenotype, harbouring a heterozygous mutation, c.1635C>A; p.Ser545Arg, in the OPA1 gene. Reprogramming factors Oct3/4, Sox2, Klf4, and c-Myc were delivered using Sendai virus.

Establishment of a human iPSC line (IISHDOi001-A) from a patient with McArdle disease.

Human iPSC line IISHDOi001-A was generated from fibroblasts of a patient with McArdle disease harbouring the mutation, c.148C>T; p.Arg50Ter, in the PYGM gene. Reprogramming factors Oct3/4, Sox2, Klf4, and c-Myc were delivered using Sendai virus.

Generation of a human iPSC line from a patient with Leigh syndrome.

Human iPSC line LND554SV.3 was generated from heteroplasmic fibroblasts of a patient with Leigh syndrome carrying a mutation in the MT-ND5 gene (m.13513GNA; p.D393N). Reprogramming factors Oct3/4, Sox2, Klf4,and cMyc were delivered using a non-integrative methodology that involves the use of Sendai virus.

Generation of a human control iPSC line with a European mitochondrial haplogroup U background.

Human iPSC line N44SV.5 was generated from primary normal human dermal fibroblasts belonging to the European mitochondrial haplogroup U. For this purpose, reprogramming factors Oct3/4, Sox2, Klf4, and cMyc were delivered using a non-integrative methodology that involves the use of Sendai virus.

Generation of a human iPSC line from a patient with a defect of intergenomic communication.

Human iPSC line PG64SV.2 was generated from fibroblasts of a patient with a defect of intergenomic communication. This patient harbored a homozygous mutation (c.2243G>C; p.Trp748Ser) in the gene encoding the catalytic subunit of the mitochondrial DNA polymerase gamma gene (POLG). Reprogramming factors Oct3/4, Sox2, Klf4, and cMyc were delivered using a non integrative methodology that involves the use of Sendai virus.

Generation of a human iPSC line from a patient with a mitochondrial encephalopathy due to mutations in the GFM1 gene.

Human iPSC line GFM1SV.25 was generated from fibroblasts of a child with a severe mitochondrial encephalopathy associated with mutations in the GFM1 gene, encoding the mitochondrial translation elongation factor G1. Reprogramming factors OCT3/4, SOX2, CMYC and KLF4 were delivered using a non integrative methodology that involves the use of Sendai virus.

Generation of a human iPSC line from a patient with an optic atrophy 'plus' phenotype due to a mutation in the OPA1 gene.

Human iPSC line Oex2054SV.4 was generated from fibroblasts of a patient with an optic atrophy 'plus' phenotype associated with a heterozygous mutation in the OPA1 gene. Reprogramming factors OCT3/4, SOX2, CMYC and KLF4 were delivered using a non-integrative methodology that involves the use of Sendai virus.

Whole-exome sequencing identifies a variant of the mitochondrial MT-ND1 gene associated with epileptic encephalopathy: west syndrome evolving to Lennox-Gastaut syndrome.

We describe a West syndrome (WS) patient with unidentified etiology that evolved to Lennox-Gastaut syndrome. The mitochondrial respiratory chain of the patient showed a simple complex I deficiency in fibroblasts. Whole-exome sequencing (WES) uncovered two heterozygous mutations in NDUFV2 gene that were reassigned to a pseudogene. With the WES data, it was possible to obtain whole mitochondrial DNA sequencing and to identify a heteroplasmic variant in the MT-ND1 (MTND1) gene (m.3946G>A, p.E214K). The expression of the gene in patient fibroblasts was not affected but the protein level was significantly reduced, suggesting that protein stability was affected by this mutation. The lower protein level also affected assembly of complex I and supercomplexes (I/III2 /IV and I/III2 ), leading to complex I deficiency. While ATP levels at steady state under stress conditions were not affected, the amount of ROS produced by complex I was significantly increased.

Cardiopatías congénitas y cromosomopatías en vida fetal: ¿siempre cariotipo? / Congenital heart defects and chromosomal anomalies in fetal life: should a chromosomal study always be offered?

Objetivos: Analizar la relación entre las cardiopatías congénitas (CC) y las cromosomopatías en vida fetal. Método: Estudio retrospectivo realizado en un centro terciario de referencia. Seleccionamos las CC diagnosticadas prenatalmente entre 1990 y 2011, con verificación posnatal del diagnóstico y con información disponible del cariotipo. La recomendación de realizar técnica invasiva prenatal para estudio del cariotipo dependió del tipo de CC y de la existencia de otros factores de riesgo de cromosomopatía. Resultados: Se analizaron 1.384 CC. El cariotipo se estudió prenatalmente en 848 (61,3%) y en el resto o se estudió posnatalmente (172; 12,4%) o se excluyó clínicamente la presencia de cromosomopatía por la ausencia de marcadores clínicos indicativos de aquella (364; 26,3%). Existía una cromosomopatía en 363 CC (26,2%). El diagnóstico fue prenatal en 324 (89,3%) y posnatal en 39 (10,7%). En estos casos no se realizó el estudio prenatal del cariotipo principalmente por la negativa de los padres (n = 28). La CC que mostró mayor asociación con cromosomopatías fue el canal aurículo-ventricular (66,7%). Esta asociación fue nula en algunas CC como la transposición de grandes arterias o el ventrículo único. Lo mismo sucedió en la atresia tricúspide aislada y en los síndromes de heterotaxia sin anomalías ajenas a las que forman parte del síndrome. Conclusiones: Aun siendo enormemente relevante la información del cariotipo en los fetos con CC para la toma de decisiones de los padres y el pronóstico del paciente, la recomendación de dicho estudio ha de individualizarse según las características de cada caso, pudiendo evitarse los riesgos de la técnica invasiva diagnóstica en muchos casos(AU)

Objectives: To assess the relationship between congenital heart defects (CHD) and chromosomal abnormalities in fetal life. Methods: This is a retrospective study undertaken at a tertiary care referral center. Our database was queried for cases of CHD prenatally diagnosed between 1990 and 2011, with postnatal diagnostic verification, as well as information available as regards the karyotype. The recommendation for performing fetal invasive procedures relied upon the type of CHD and the presence of associated high-risk factors of chromosomal disease. Results: A total of 1,384 CHD were retrieved and analyzed. The karyotype was studied prenatally in 848 (61.3%) and in the rest was either studied postnatally (172, 12.4%) or the presence of chromosomal disease was clinically ruled out given the absence of suggestive clinical markers (364, 26.3%). Chromosomal defects were diagnosed in 363 CHD (26.2%). The diagnosis was made prenatally in 324 (89.3%), and after birth in 39 (10.7%). In most of these cases (n = 28) the parents refused fetal invasive testing. We found that atrioventricular septal defect was the CHD most associated with chromosomal abnormalities (66.7%). On the contrary, we did not observe any chromosomal defect in CHD, such as transposition of large arteries or single ventricle. Similarly, there was no abnormal karyotype in isolated tricuspid atresia or in heterotaxy syndromes presenting without anomalies other than those typically included in the disease. Conclusions: Karyotype analysis is highly relevant in fetuses with CHD, given its impact in the parental decision-making process and patient outcome. Nevertheless, the recommendation of performing fetal invasive testing should be based on the individual characteristics of any given case, and in many cases the risks associated with the invasive procedure could be avoidable(AU)

Noninvasive fetal sex determination in maternal plasma: a prospective feasibility study.

PURPOSE: To prospectively validate a protocol for noninvasive fetal sex determination in maternal plasma and demonstrate its applicability to clinical practice. METHODS: Peripheral blood from 404 pregnant women undergoing prenatal invasive testing was collected from 6 to 23 weeks of gestation. Real-time PCR was performed for the SRY gene and multicopy DYS14 marker sequence located within the TSPY gene by the TaqMan minor groove binder probe assay as a first-line test. Owing to a false-positive result, amplification of repetitive motifs of the DAZ gene region was also tested as a second-line test performed in the last 232 patients enrolled in our series. A diagnostic algorithm was designed using a combination of these three markers. Fetal gender determined by noninvasive prenatal diagnosis (NIPD) was compared with that diagnosed by quantitative fluorescent PCR after invasive testing or ultrasound. RESULTS: A single false-positive result was obtained in the first 172 pregnancies. Reporting criteria were modified in the subsequent 232 pregnancies, giving an overall sensitivity and specificity of 100% (95% CI 99.8-100%) and 99.5% (95% CI 98.1-100%), respectively. Pregnancy outcome was obtained in all cases, including 221 male-bearing and 183 female-bearing pregnancies. CONCLUSION: NIPD for fetal sex determination in maternal plasma is highly accurate and clinically applicable if robust reporting criteria are applied.