Prenatal diagnosis of X-linked myopathy associated with a VMA21 gene mutation afforded through a novel targeted exome sequencing strategy applied in fetuses with abnormal ultrasound findings.

Fetal malformations detected through routine prenatal ultrasound examination comprise a heterogeneous group potentially associated with genetic disorders where the underlying cause is difficult to establish. We present the prenatal diagnosis of a rare X-linked myopathy involving a new VMA21 gene mutation, detected through a novel prenatal exome sequencing-based approach.

The APOE E4 Allele Confers Increased Risk of Ischemic Stroke Among Greek Carriers.

BACKGROUND: Although several studies in various countries have indicated that the presence of the E4 allele of the apolipoprotein-E (APOE) gene is a risk factor for ischemic cerebrovascular disease, the strength of this association still remains a matter of debate. OBJECTIVES: The aim of the study was to determine the frequency of the APOE E4 allele and various other gene polymorphisms in in a well-characterized sample of Greek patients and to evaluate the potential associations with the risk of ischemic stroke (IS) and coronary heart disease (CHD). MATERIAL AND METHODS: A total of nine gene variants/polymorphisms - F5 (Leiden - R5 06Q, rs6025), F2 (20210G > A, rs1799963), F13A1 (V34L, rs5985), MTHFR (677C > T - A222V, rs1801133), MTHFR (1298A > C - E429A, rs1801131), FGB (-455G > A -c.-463G > A; rs1800790), SERPINE1 (PAI14G/5G - rs1799889), ACE (ACE I/D, rs1799752), ITGB3 (GPIIIa L33P, rs5918) and the APOE E2/E3/E4 alleles (rs7412, rs429358) - were genotyped in 200 newly diagnosed ischemic stroke (IS) patients, 165 patients with ischemic coronary heart disease (CHD) and 159 controls with no cerebroor cardiovascular disease (non-CVD). A statistical analysis was performed using univariate and multivariate logistic regression models. RESULTS: No significant association was found regarding most gene polymorphisms and the presence of IS or CHD in the patient cohort. However, the APOE E4 allele frequency was significantly higher (p = 0.02) among patients with ischemic stroke (IS) or IS + CHD (12.7%) when compared to the controls (5.1%). More accurately, E4 carriers had 2.66 and 2.71 times greater likelihood of IS or IS + CHD than non-carriers, respectively (OR = 2.66, 95% CI 1.39-5.07, OR = 2.71, 95% CI 0.98-7.48). CONCLUSIONS: In contrast to some previous studies, these results support the role of the APOE E4 allele as an independent risk factor for ischemic stroke and ischemic coronary heart disease among Greek patients.

First applications of a targeted exome sequencing approach in fetuses with ultrasound abnormalities reveals an important fraction of cases with associated gene defects.

Background. Fetal malformations and other structural abnormalities are relatively frequent findings in the course of routine prenatal ultrasonographic examination. Due to their considerable genetic and clinical heterogeneity, the underlying genetic cause is often elusive and the resulting inability to provide a precise diagnosis precludes proper reproductive and fetal risk assessment. We report the development and first applications of an expanded exome sequencing-based test, coupled to a bioinformatics-driven prioritization algorithm, targeting gene disorders presenting with abnormal prenatal ultrasound findings. Methods. We applied the testing strategy to14 euploid fetuses, from 11 on-going pregnancies and three products of abortion, all with various abnormalities or malformations detected through prenatal ultrasound examination. Whole exome sequencing (WES) was followed by variant prioritization, utilizing a custom analysis pipeline (Fetalis algorithm), targeting 758 genes associated with genetic disorders which may present with abnormal fetal ultrasound findings. Results. A definitive or highly-likely diagnosis was made in 6 of 14 cases (43%), of which 3 were abortuses (Ellis-van Creveld syndrome, Ehlers-Danlos syndrome and Nemaline myopathy 2) and 3 involved on-going pregnancies (Citrullinemia, Noonan syndrome, PROKR2-related Kallmann syndrome). In the remaining eight on-going pregnancy cases (57%), a ZIC1 variant of unknown clinical significance was detected in one case, while in seven cases testing did not reveal any pathogenic variant(s). Pregnancies were followed-up to birth, resulting in one neonate harboring the PROKR2 mutation, presenting with isolated minor structural cardiac abnormalities, and in seven apparently healthy neonates. Discussion. The expanded targeted exome sequencing-based approach described herein (Fetalis), provides strong evidence suggesting a definite and beneficial increase in our diagnostic capabilities in prenatal diagnosis of otherwise chromosomally balanced fetuses with troubling ultrasound abnormalities. Furthermore, the proposed targeted exome sequencing strategy, designed primarily as a diagnostic rather than a research discovery tool, overcomes many of the problems and limitations associated with clinical wide-scale WES testing in a prenatal setting.

Dilemmas in Prenatal Chromosomal Diagnosis Revealed Through a Single Center's 30 Years' Experience and 90,000 Cases.

INTRODUCTION: The aim of this article is to provide a perspective of prenatal chromosomal diagnosis (PCD) derived from a single center's evolving experience from ∼90,000 consecutive prenatal cases and to highlight important issues and current dilemmas. MATERIALS AND METHODS: Prenatal cases in this study (1985-2013) were referred for various indications, and PCD was performed by standard karyotype in 84,255 cases, multiplex ligation-dependent probe amplification (MLPA) panel in 3,010 cases and standalone array comparative genomic hybridization (aCGH) in 3,122 cases. RESULTS: Classic karyotype revealed 1.7 and 7.9% of pathological cases in amniotic fluid and CVS samples, respectively, with common aneuploidies accounting for 59.6 and 64.3% of the total abnormal. Molecular approaches increased the diagnostic yield by 0.6% for MLPA and 1.6% for aCGH, uncovering pathogenic chromosomal abnormalities undetectable by karyotype analysis. CONCLUSIONS: Current molecular diagnostic capabilities and the recent introduction of noninvasive prenatal testing (NIPT) point to one current major dilemma in PCD, with serious implications in genetic counseling, relating on the one hand to reaping the benefits from the high detection rate afforded through aCGH but accepting an invasive risk, and on the other hand, offering a lower detection rate practically only for Down syndrome, with minimal invasive risk.

A familial case of Muenke syndrome. Diverse expressivity of the FGFR3 Pro252Arg mutation--case report and review of the literature.

Muenke is a fibroblast growth factor receptor 3 (FGFR-3)-associated syndrome, which was first described in late 1990 s. Muenke syndrome is an autosomal dominant disorder characterized mainly by coronal suture craniosynostosis, hearing impairment and intellectual disability. The syndrome is defined molecularly by a unique point mutation c.749C > G in exon 7 of the FGFR3 gene which results to an amino acid substitution p.Pro250Arg of the protein product. Despite the fact that the mutation rate at this nucleotide is one of the most frequently described in human genome, few Muenke familial case reports are published in current literature. We describe individuals among three generations of a Greek family who are carriers of the same mutation. Medical record and physical examination of family members present a wide spectrum of clinical manifestations. In particular, a 38-year-old woman and her father appear milder clinical findings regarding craniofacial characteristics compared to her uncle and newborn female child. This familial case illustrates the variable expressivity of Muenke syndrome in association with an identical gene mutation.

Routine application of a novel MLPA-based first-line screening test uncovers clinically relevant copy number aberrations in haematological malignancies undetectable by conventional cytogenetics.

OBJECTIVE: The presence of numerical and/or structural chromosomal abnormalities is a frequent finding in clonal hematopoietic malignant disease, typically diagnosed through routine karyotyping and/or fluorescent in situ hybridization (FISH) analysis. Recently, the application of array comparative genomic hybridization (aCGH) has uncovered many new cryptic genomic copy number imbalances, most of which are now recognized as clinically useful markers of haematological malignancies. In view of the limitations of both FISH and aCGH techniques, in terms of their routine application as a first line screening test, we designed a new multiple ligation-dependent probe amplification (MLPA) probemix for use in addition to classic karyotype analysis. METHODS: A novel MLPA probemix was developed to interrogate copy number changes involving chromosomal regions: 2p23-24 (MYCN, ALK), 5q32-34 (MIR145A, EBF1, MIR146A), 6q21-27, 7p12.2 (IKZF1), 7q21-36, 8q24.21 (MYC), 9p24 (JAK2 V617F point mutation), 9p21.3 (CDKN2A/2B), 9p13.2 (PAX5), 10q23 (PTEN), 11q22.3 (ATM), 12p13.2 (ETV6), 13q14 (RB1, MIR15A, DLEU2, DLEU1), 17p13.1 (TP53), and 21q22.1 (RUNX1/AML1) and was applied to DNA extracted from 313 consecutive bone marrow patient samples, referred for routine karyotype analysis. RESULTS: More than half of the samples originated from newly investigated patients. We discovered clinically relevant genomic aberrations, involving a total of 24 patients (8%) all with a normal karyotype, which would have remained undiagnosed. DISCUSSION: Our data clearly indicate that routine application of this MLPA screening panel, as an adjunct to karyotype analysis, provides a sensitive, robust, rapid and low-cost approach for uncovering clinically important genomic abnormalities, which would have otherwise remained undetected.

A novel ß(0)-thalassemia frameshift mutation: [HBB:c.216delT].

A 33-year-old adult male of Greek ethnicity, with hematological indices suggesting ß(0)-thalassemia (ß(0)-thal) trait, was investigated for HBB gene mutations in the course of preparation for preimplantation genetic diagnosis (PGD). Application of a routine diagnostic protocol, consisting of sequence analysis of the HBB gene, coupled to multiplex ligation-dependent probe amplification (MLPA), identified a single nucleotide deletion (-T) at codon 72 [HBB: c.216delT], leading to a novel pathogenic frameshift and protein-truncating ß(0)-thal mutation (p.Phe72LeufsX18).

Uncovering recurrent microdeletion syndromes and subtelomeric deletions/duplications through non-selective application of a MLPA-based extended prenatal panel in routine prenatal diagnosis.

OBJECTIVE: To present the application of multiplex ligation-dependent probe amplification (MLPA)-based screening approach in 1550 typical prenatal cases, for the simultaneous targeted detection of 23 recurrent microdeletion syndromes as well as subtelomeric copy number assessment for all chromosomes and discuss the implications in routine prenatal chromosomal diagnosis (PCD). METHODS: Following amniocentesis or chorionic villus sampling, samples were processed for routine karyotype analysis while DNA was extracted in parallel for MLPA analysis. When necessary, parental samples were analyzed to determine the inheritance of the detected aberrations. RESULTS: This panel has been applied since 2006 in 1550 prenatal samples, referred for routine karyotype analysis, with (16.1%) or without (77.7%) ultrasound (US) findings. We identified eight fetuses with pathological genomic abnormalities (approximately 1 in 193), five of which had as sole indication advanced maternal age (1 in 240). In two cases an abnormality was suspected from karyotype analysis, while the remaining six cases would have otherwise remained totally undetected. CONCLUSIONS: Our data represent the largest published series involving this type of genomic analysis in routine prenatal diagnosis, without indication bias. The panel increases significantly the diagnostic yield of conventional PCD and does not pose interpretation problems.

Successful hematopoietic stem cell transplantation in 2 children with X-linked chronic granulomatous disease from their unaffected HLA-identical siblings selected using preimplantation genetic diagnosis combined with HLA typing.

We report 2 children with X-linked chronic granulomatous disease (X-CGD) who underwent hematopoietic stem cell transplantation (HSCT) using grafts from their siblings selected before implantation to be both unaffected and HLA-matched donors. Preimplantation genetic diagnosis (PGD) along with HLA-typing were performed on preimplantation embryos by single-cell multiplex polymerase chain reaction using informative short tandem repeat markers in the HLA locus together with the gene region containing the mutations. Two singleton pregnancies resulted from the intrauterine transfer of selected embryos; these developed to term, producing 1 healthy female and 1 X-CGD carrier female, which are HLA-identical siblings to the 2 affected children. Combined grafts of umbilical cord blood (UCB) and bone marrow (BM) stem cells were administered to the recipients after myeloablative (MA) conditioning at the ages of 4.5 years and 4 years, respectively. Both patients are well, with complete donor hematopoietic and immunologic reconstitution, at 18 and 13 months posttransplantation, respectively. This report demonstrates that HSCT with HLA-matched sibling donors created by PGD/HLA typing of in vitro fertilized embryos is a realistic therapeutic option and should be presented as such to families with children who require a non-urgent HSCT but lack an HLA-genoidentical donor.

Birth of a healthy histocompatible sibling following preimplantation genetic diagnosis for chronic granulomatous disease at the blastocyst stage coupled to HLA typing.

OBJECTIVE: To perform preimplantation genetic diagnosis (PGD) for chronic granulomatous disease with simultaneous HLA typing in a family case with an affected male child, with the aim of selecting unaffected and HLA-matched embryos to act as donors for hematopoietic stem cell transplantation from umbilical cord blood. METHODS: A flexible, indirect HLA haplotyping protocol, based on single-cell multiplex PCR analysis of multiple polymorphic short tandem repeat (STR) markers within the human HLA complex, was optimized for the simultaneous amplification of the informative STR markers together with the gp91-phox gene region containing the mutation and the sexing marker amelogenin. Detection of the c.469C>T disease mutation was performed by minisequencing. Biopsy was performed at the blastocyst stage on day 5 by removal of trophectoderm cells. RESULTS: A total of 11 blastocysts were biopsied on day 5 and a successful result for the informative STR markers and for the mutation detection was obtained for all 11 blastocyst samples. Two healthy and HLA-matched embryos were identified and transferred resulting in a singleton pregnancy. The results of the PGD were confirmed following standard prenatal diagnosis, and cord blood hemopoietic stem cells were obtained from the newborn for subsequent transplantation into the affected sibling. CONCLUSIONS: This study demonstrates the feasibility and first successful application of this complex PGD approach as a therapeutic option in families with children affected with X-linked chronic granulomatous disease.

CFTR DeltaF508 mutation detection from dried blood samples in the first trimester of pregnancy: a possible routine prenatal screening strategy for cystic fibrosis?

OBJECTIVE: The implementation and evaluation of a proposed wide-scale prenatal screening strategy, based on DNA isolated from dried blood spots in the first trimester of pregnancy, for the early detection of pregnancies at risk for cystic fibrosis (CF). METHODS: The screening was performed in conjunction with routine biochemical marker screening for Down's syndrome risk in the first trimester of pregnancy. DNA was isolated from 1,233 dried blood spots and analyzed for the presence of the CF transmembrane regulator DeltaF508 mutation. Women carriers were offered and accepted the option for additional full testing of their partners in order to assess the risk for the fetus. RESULTS: All 1,233 samples were successfully analyzed, identifying 23 DeltaF508 carriers, corresponding to a DeltaF508 carrier rate of approximately 1/55 (1.8%). All partners of the women carriers were further tested without revealing any need for further prenatal testing in this group. CONCLUSIONS: This study reveals the relatively high frequency of the DeltaF508 CF mutation in the Greek population. More importantly, we demonstrate that the proposed prenatal screening strategy, based on the ease and cost-effectiveness of the analysis for the detection of a single common mutation, can be considered as a feasible and practical approach for wide-scale prenatal screening for CF, following the sequential model. It is applied early on in pregnancy, allowing for the timely management of families at risk for the corresponding genetic disorders. Finally, it can easily be extended to include screening for other common genetic disorders in specific population groups.

Acute myelogenous leukemia with tetrasomy 8 is a disease with a poor prognosis.

Tetrasomy 8 is an extremely rare chromosome abnormality, one that has been reported in only a few cases with myeloid malignancies. The majority of reported cases consist of acute myelogenous leukemias (AML) of monocytic lineage. In slightly more than half of the patients, tetrasomy 8 was the single cytogenetic abnormality. Fluorescence in situ hybridization revealed tetrasomy 8 and trisomy 8 concurrently in all but one of the bone marrow samples. The clonal relationship between trisomy 8 and tetrasomy 8 in these cases remains to be clarified. Patients with tetrasomy 8 have a poor prognosis, and only 1 out of 33 patients was free of disease 3 years after autologous bone marrow transplantation. Here, we report the case of a 25-year-old female patient with monocytic leukemia and tetrasomy 8.

Expression of recombination activating genes-1 and-2 immunoglobulin heavy chain gene rearrangements in acute myeloid leukemia: evaluation of biological and clinical significance in a series of 76 uniformly treated patients and review of the literature.

BACKGROUND AND OBJECTIVES: Early lymphoid differentiation is characterized by antigen receptor gene rearrangements; the rearrangement process is governed by two lymphoid-specific genes, RAG (recombinase activating gene)-1 and -2. The available data on the incidence and prognostic significance of clonal immunoglobulin heavy chain (IgH) gene rearrangements in acute myeloid leukemia (AML) are rather contradictory. The aim of this study was to evaluate the incidence and prognostic significance of RAG-1 and -2 mRNA transcripts and clonal IgH gene rearrangements in a cohort of uniformly treated AML patients; the available literature is also reviewed. DESIGN AND METHODS: The study was performed on 76 AML patients, newly diagnosed between August 1996 and November 1999. RAG-1/-2 gene expression was analyzed by a reverse transcriptase polymerase chain reaction technique and IgH gene rearrangements were detected with variable region (VH) family-specific and consensus framework region (FWR)-2 and/or-3 primers. Statistical associations were explored between IgH monoclonality/ RAG mRNA expression and: (i) age, gender, FAB subtype, immunophenotype, cytogenetic risk groups; (ii) response variables (response/relapse incidence, survival). RESULTS: In total, 38/75 samples (50.6%) were RAG-1 and/or -2 positive; 30/76 samples (39.5%) carried clonal IgH genes, whereas 13/30 IgH-positive samples (43.3%) were RAG-1/2-negative. Significant associations were detected only for RAG-2 positivity and unfavorable karyotype and IgH monoclonality and FAB subtypes M4/ M5; no association was identified with response outcome and survival. INTERPRETATION AND CONCLUSIONS: Lymphoid-specific molecular markers are detected in a significant proportion of AML patients, regardless of differentiation status (assessed morphologically/ immunophenotypically); however, in our experience, they do not seem to constitute an adverse prognostic factor.

Malignancy: Molecular Demonstration of BCR/ABL Fusion in a Patient with Chronic Myelogenous Leukemia with Basophilia Carrying a Variant t(16;22) (q24;q11) Philadelphia Chromosome.

We report a patient with chronic myelogenous leukemia in chronic phase and basophilia which was found to carry a simple variant t(16;22) (q24;q11) Philadelphia (Ph) chromosome in unstimulated bone marrow mononuclear cells. Molecular analysis of peripheral blood and bone marrow mononuclear cells demonstrated the presence of a bcr-abl chimeric mRNA transcript of the b(3) -a(2) type. These findings confirm that band 9q34 participates in the formation of all Ph chromosomes, either standard or variant, even when this is not detectable by conventional cytogenetics. The available literature concerning variant Philadelphia translocations is also reviewed.