Post-mortem diagnosis of Pompe disease by exome sequencing in a Moroccan family: a case report.

BACKGROUND: Pompe disease is an autosomal recessive lysosomal storage disorder characterized by progressive myopathy with proximal muscle weakness, respiratory muscle dysfunction, and cardiomyopathy. Its prevalence ranges between 1/9000 and 1/40,000. It is caused by compound heterozygous or homozygous mutations in the GAA gene, which encodes for the lysosomal enzyme alpha-glucosidase, required for the degrading of lysosomal glycogen. CASE PRESENTATION: In this study, we report the case of a Moroccan consanguineous family with hypertrophic cardiomyopathy and sudden cardiac deaths at an early age; our patient was a 7-month-old Moroccan girl. Whole exome sequencing identified the deleterious homozygous mutation c.236_246delCCACACAGTGC (p.Pro79ArgfsX13) of GAA gene leading to a post-mortem diagnosis of Pompe disease. CONCLUSION: The identification of the genetic substrate in our patient, the daughter, confirmed the clinical diagnosis of Pompe disease and allowed us to provide appropriate genetic counseling to the family for future pregnancies.

A novel non sense mutation in WDR62 causes autosomal recessive primary microcephaly: a case report.

BACKGROUND: Autosomal recessive primary microcephaly (MCPH) is a rare genetically heterogeneous disorder of neurogenic brain development characterized by a reduced head circumference at birth with no remarkable anomalies of brain architecture and variable degrees of intellectual impairment. Clinical and genetic heterogeneity in genetic disorders represent a major diagnostic challenge. CASE PRESENTATION: Two patients, 11 and 9 years old, born from consanguineous parents, were referred to the department of medical genetics at the National Institute of Health in Rabat. The diagnosis of MCPH was made, based on reduced head circumference without brain architecture abnormalities. The two patients were subject to the whole-exome sequencing, which allowed to diagnose a novel homozygous mutation c.1027C > T; p.Gln343* in exon 8 of WDR62, a gene already known to be related to MCPH. Sanger sequencing confirmed the segregation of the mutation in the family. CONCLUSION: Our data expends the spectrum of mutations in WDR62 gene, proves the efficiency and cost-effectiveness of whole exome sequencing for the molecular diagnosis of genetically heterogeneous disorders such MCPH. Exome sequencing led to the rapid and cost-effective identification of a novel homozygous mutation in WDR62 gene, thereby facilitating genetic counseling.

Next Generation Sequencing identifies mutations in GNPTG gene as a cause of familial form of scleroderma-like disease.

BACKGROUND: Scleroderma is a multisystem disease, characterized by fibrosis of skin and internal organs, immune dysregulation, and vasculopathy. The etiology of the disease remains unknown, but it is likely multifactorial. However, the genetic basis for this condition is defined by multiple genes that have only modest effect on disease susceptibility. METHODS: Three Moroccan siblings, born from non-consanguineous Moroccan healthy parents were referred for genetic evaluation of familial scleroderma. Whole Exome Sequencing was performed in the proband and his parents, in addition to Sanger sequencing that was carried out to confirm the results obtained. RESULTS: Mutation analysis showed two compound heterozygous mutations c.196C>T in exon 4 and c.635_636delTT in exon 9 of GNPTG gene. Sanger sequencing confirmed these mutations in the affected patient and demonstrated that their parents are heterozygous carriers. CONCLUSION: Our findings expand the mutation spectrum of the GNPTG gene and extend the knowledge of the phenotype-genotype correlation of Mucolipidosis Type III gamma. This report also highlights the diagnostic utility of Next Generation Sequencing particularly when the clinical presentation did not point to specific genes.

Identification of two novel SH3PXD2B gene mutations in Frank-Ter Haar syndrome by exome sequencing: Case report and review of the literature.

BACKGROUND: Frank-Ter Haar syndrome (FTHS) is an autosomal-recessive disorder characterized by skeletal, cardio-vascular, and eye abnormalities, such as increased intraocular pressure, prominent eyes, and hypertelorism. The most common underlying genetic defect in Frank-Ter Haar syndrome appears to be due to mutations in the SH3PXD2B gene on chromosome 5q35.1. Until now, only six mutations in SH3PXD2B gene have been identified. A genetic heterogeneity of FTHS was suggested in previous studies. DESIGN: FTHS was suspected clinically in a girl of 2years old, born from non-consanguineous Moroccan healthy parents. The patient had been referred to a medical genetics outpatient clinic for dysmorphic facial features. Whole Exome Sequencing (WES) was performed in the patient and her parents, in addition to Sanger sequencing that was carried out to confirm the results. RESULTS: We report the first description of a Moroccan FTHS patient with two novel compound heterozygous mutations c.806G>A; p.Trp269* (maternal allele) and c.892delC; p.Asp299Thrfs*44 (paternal allele) in the SH3PXD2B gene. Sanger sequencing confirmed this mutation in the affected girl and demonstrated that her parents carry this mutation in heterozygous state. CONCLUSION: Our results confirm the clinical diagnosis of FTHS in this reported family and contribute to expand the mutational spectrum of this rare disease. Our study shows also, that exome sequencing is a powerful and a cost-effective tool for the diagnosis of a supposed genetically heterogeneous disorder such FTHS.

Expression profile of genes coding for DNA repair in human oocytes using pangenomic microarrays, with a special focus on ROS linked decays.

PURPOSE: To determine the level of expression for mRNAs that regulate DNA repair activity in oocytes at the germinal vesicle (GV) stage. Reactive oxygen species (ROS) have been shown to play a major role in the appearance of deleterious DNA decays, and this study focuses on the repair of damage linked to decay caused by the action of ROS. The oocyte needs a mechanism for repairing DNA decays in the early preimplantation embryo before the onset of genomic activation, since in the absence of repair, residual DNA damage would lead to either apoptosis or tolerance. Tolerance of DNA damage is a source of potential mutations. METHOD: GV oocytes were selected for this study, both for the ethical reason that they are unsuitable for patient treatment, and because no transcription takes place during the period from GV to MII and then prior to genomic activation. The GV oocyte is therefore a good model for looking at DNA during the first cleavages of early preimplantation development. Six cohorts of GV oocytes were pooled for extraction of mRNA; the DNA was analysed using Affimetrix HG-UG133 Plus 2, containing 54,675 probe sets; spike and housekeeping genes were also added as internal controls. RESULTS: In GV oocytes, DNA repair pathways for oxidized bases are redundant. One step repair procedure (OSR), BER (base excision repair), MMR (mismatch repair) and NER (Nucleotide excision repair) are present. All the recognition proteins are also present. The chromatin assembly factors necessary for the maintenance of genomic stability are highly expressed. CONCLUSION: Gene expression analysis shows that the oocyte does not allow a high level of tolerance for DNA decays. This regulatory mechanism should avoid transmitting mutations into the next generation.

APEX/Ref-1 (apurinic/apyrimidic endonuclease DNA-repair gene) expression in human and ascidian (Ciona intestinalis) gametes and embryos.

In recent years, the impact of sperm DNA damage on fertility has become an important issue. The different technologies developed to check sperm DNA fragmentation lead to the same conclusion: DNA damage negatively impacts upon reproductive processes. Oocyte DNA repair capacity is one of the cues to understanding embryo developmental arrest. APEX/Ref-1 (apurinic/apyrimidic endonuclease) is an enzyme involved in the DNA base excision repair pathway removing the abasic sites, the most common DNA decays. In humans, APEX has a multifunctional role, including the control of the redox status of transcription factors. RT-PCR allowed us to detect human APEX transcripts in oocytes, spermatozoa and preimplantation blocked embryos. In parallel, a comparative study on sea squirt Ciona intestinalis (ascidian) indicated that APEX transcripts are clearly detectable in oocytes and embryos until the larva stage, but not in spermatozoa, suggesting the appearance of the paternal contribution to DNA repair during development having arisen only late in Vertebrate evolution. Of additional phylogenetic significance is the observation that sea squirt APEX appears to lack redox transcriptional activity.

Genetic expression of monocarboxylate transporters during human and murine oocyte maturation and early embryonic development.

During the early preimplantationes of human embryos, pyruvate and lactate, but not glucose, are the preferred energy substrates. Transport of these monocarboxylates is mediated, in mammalian cells, by a family of transporters, designated as monocarboxylate transporters (MCTs). Human and mouse genetic expression of MCT members 1, 2, 3, 4 and basigin, a chaperone protein of MCT1 and MCT4, was qualitatively analysed using the reverse transcription nested polymerase chain reaction (RT-nested PCR) in immature oocytes (germinal vesicle stage; GV), in non-fertilised metaphase II (MII) oocytes and in embryos from 2-cell stage to blastocysts. Transcripts encoding for MCT1 and MCT2 were present, under a polyadenylated form, in the majority of the human and mouse oocytes and early embryos. MCT3 transcripts were not detected in either human or mouse. MCT4 mRNA was not detected in human oocytes and embryos, but was present in mouse oocytes and embryos. This fact could imply differences in lactate transport and regulation of intracellular pH between human and murine early embryos. Basigin transcripts were present in mouse and human MII oocytes and preimplantation embryos, but were not detected at GV stage. However, using 3' end-specific primers in the RT reaction instead of Oligo(dT)12-18 primers, transcripts encoding for this protein were then detected at GV stage in both species. This result suggests that a regulated polyadenylation process occurs during oocyte maturation for these transcripts. Thus, basigin mRNA can be considered as a marker of oocyte cytoplasmic maturation in human and mouse species.