Molecular characterization of novel and rare DNA variants in patients with galactosemia.

Introduction: Galactosemia is an inherited disorder caused by mutations in the three genes that encode enzymes implicated in galactose catabolism. Currently, the only available treatment for galactosemia is life-long dietary restriction of galactose/lactose, and despite treatment, it might result in long-term complications. Methods: Here, we present five cases of newborn patients with elevated galactose levels, identified in the context of the newborn screening program. Genetic analysis concerned a next generation sequencing (NGS) methodology covering the exons and adjacent splice regions of the GALT, GALK1, and GALE genes. Results: Our approach led to the identification of eight rare nonsynonymous DNA variants. Four of these variants, namely, p.Arg204Gln and p.Met298Ile in GALT, p.Arg68Leu in GALK1, and p.Ala180Thr in GALE, were already recorded in relevant databases, yet their clinical significance is uncertain. The other four variants, namely, p.Phe245Leu in GALT, p.Gly193Glu in GALK1, and p.Ile266Leu and p.Ala216Thr in the GALE gene, were novel. In silico analysis of the possible effect of these variants in terms of protein function and stability was performed using a series of bioinformatics tools, followed by visualization of the substituted amino acids within the protein molecule. The analysis revealed a deleterious and/or destabilizing effect for all the variants, supported by multiple tools in each case. Discussion: These results, given the extreme rarity of the variants and the specific phenotype of the respective cases, support a pathogenic effect for each individual variant. Altogether, our study shows that targeted NGS methodologies may offer a time- and cost-effective approach for the genetic investigation of galactosemia and can assist in elucidating the complex genetic background of this disorder.

Deregulation of Autophagy and Apoptosis in Patients with Myelodysplastic Syndromes: Implications for Disease Development and Progression.

(1) Background: Myelodysplastic neoplasms (MDSs) consist of a group of blood malignancies with a complex biological background. In this context, we investigated the role of autophagy and apoptosis in the pathogenesis and progression of MDSs. (2) Methods: To address this issue, we performed a systematic expression analysis on a total of 84 genes in patients with different types of MDSs (low/high risk of malignancy) versus healthy individuals. Furthermore, real-time quantitative PCR (qRT-PCR) was used to validate significantly upregulated or downregulated genes in a separate cohort of MDS patients and healthy controls. (3) Results: MDS patients were characterized by lower expression levels for a large series of genes involved in both processes compared to healthy individuals. Of importance, deregulation was more pronounced in patients with higher-risk MDS. Results from the qRT-PCR experiments displayed a high level of concordance with the PCR array, strengthening the relevance of our findings. (4) Conclusions: Our results indicate a clear effect of autophagy and apoptosis on MDS development, which becomes more pronounced as the disease progresses. The results from the present study are expected to assist in our understanding of the biological background of MDSs as well as in the identification of novel therapeutic targets.

Induction of MMP-3 and MMP-9 expression during Helicobacter pylori infection via MAPK signaling pathways.

BACKGROUND AND AIMS: Helicobacter pylori (H. pylori)-induced gastric pathology involves remodeling of extracellular matrix mediated by aberrant activity of matrix metalloproteinases (MMPs). We have previously shown that in vitro H. pylori infection leads to MMP-3 and MMP-9 overexpression, associated with phosphorylation of bacterial oncoprotein CagA. We extended these findings in an in vivo model of H. pylori infection and further assessed the involvement of MAPK pathways in MMP expression. MATERIALS AND METHODS: C57BL/6 mice were infected with H. pylori strains HPARE, HPARE ΔCagA, and SS1, for 6 and 9 months. Transcriptional expression of Mmp-3 and Mmp-9 was evaluated via qPCR while respective protein levels in the gastric mucosa were determined immunohistochemically. Epithelial cell lines AGS and GES-1 were infected with H. pylori strain P12 in the presence of chemical inhibitors of JNK, ERK1/2, and p38 pathways, for 24 h. mRNA and protein expression of MMP-3 and MMP-9 were determined via qPCR and Western blot, respectively. RESULTS: We observed transcriptional activation of Mmp-3 and Mmp-9 as well as aberrant MMP-3 and MMP-9 protein expression in murine gastric tissue following H. pylori infection. CagA expression was associated with MMP upregulation, particularly during the early time points of infection. We found that inhibition of ERK1/2 resulted in reduced mRNA and protein expression of MMP-3 and MMP-9 during H. pylori infection, in both cell lines. Expressed protein levels of both MMPs were also found reduced in the presence of JNK pathway inhibitors in both cell lines. However, p38 inhibition resulted in a more complex effect, probably attributed to the accumulation of phospho-p38 and increased phospho-ERK1/2 activity due to crosstalk between MAPK pathways. CONCLUSIONS: H. pylori colonization leads to the upregulation of MMP-3 and MMP-9 in vivo, which primarily involves ERK1/2 and JNK pathways. Therefore, their inhibition may potentially offer a protective effect against gastric carcinogenesis and metastasis.

Polydactyly: Clinical and molecular manifestations.

Polydactyly is a malformation during the development of the human limb, which is characterized by the presence of more than the normal number of fingers or toes. It is considered to be one of the most common inherited hand disorders. It can be divided into two major groups: Non-syndromic polydactyly or syndromic polydactyly. According to the anatomical location of the duplicated digits, polydactyly can be generally subdivided into pre-, post-axial, and mesoaxial forms. Non-syndromic polydactyly is often inherited with an autosomal dominant trait and defects during the procedure of anterior-posterior patterning of limb development are incriminated for the final phenotype of the malformation. There are several forms of polydactyly, including hand and foot extra digit manifestations. The deformity affects upper limbs with a higher frequency than the lower, and the left foot is more often involved than the right. The treatment is always surgical. Since the clinical presentation is highly diverse, the treatment combines single or multiple surgical operations, depending on the type of polydactyly. The research attention that congenital limb deformities have recently attracted has resulted in broadening the list of isolated gene mutations associated with the disorders. Next generation sequencing technologies have contributed to the correlation of phenotype and genetic profile of the multiple polydactyly manifestations and have helped in early diagnosis and screening of most non-syndromic and syndromic disorders.

Genetics of congenital anomalies of the hand.

Congenital anomalies of the hand are malformations occurring during the development of the human limb, and present as isolated disorders or as a part of a syndrome. During the last years, molecular analysis techniques have offered increasing knowledge about the molecular basis of hand malformations. Disturbances in the signaling pathways during the development of the upper limb result in malformations of the upper extremity. At present, several genes have been identified as responsible for hand anomalies and other have been recognized as suspect genes related to them. Different and new high throughput methods have been introduced for the identification of the gene mutations. In the current editorial, we summarize concisely the current molecular status of isolated hand genetic disorders and the recent progress in molecular genetics, including the genes related to the disorder. This progress improves the knowledge of these disorders and has implications on genetic counselling and prenatal diagnosis.

Adult hypophosphatasia treated with reduced frequency of teriparatide dosing.

We report a 41-year-old man diagnosed with the adult form of hypophosphatasia (HPP) and treated for 4 years with less frequent than conventional daily doses of teriparatide (TPTD). He presented with a history of three low-energy fractures and low bone mineral density (BMD) ineffectively treated with bisphosphonate. We identified within ALPL, the gene that encodes the homodimeric "tissue-nonspecific" isoenzyme of alkaline phosphatase (ALP) and underlies HPP, a heterozygous missense mutation (c.455 G>A→R135H). Characteristic painful periarticular calcification removed at a shoulder did not recur. However, access to medical treatment with asfotase alfa (AA) was denied. After he sustained a low-energy metatarsal fracture, we administered TPTD subcutaneously "off-label" at 20 µg/d. An elbow fracture occurred two months later. Five months afterwards, due to his limited number of approved TPTD doses, TPTD treatment was extended using alternate-day dosing. Although his serum ALP activity did not increase (33-48 U/l; reference range 40-120) with 4 years of TPTD treatment, his BMD improved 15% in the lumbar spine and 6% in the femoral neck with no further fractures. Our experience represents success overcoming two prescription deadlocks; AA was denied for adult HPP, and TPTD was not to be administered daily for more than two years.

Impact of Helicobacter pylori Infection and Its Major Virulence Factor CagA on DNA Damage Repair.

Helicobacter pylori infection induces a plethora of DNA damages. Gastric epithelial cells, in order to maintain genomic integrity, require an integrous DNA damage repair (DDR) machinery, which, however, is reported to be modulated by the infection. CagA is a major H. pylori virulence factor, associated with increased risk for gastric carcinogenesis. Its pathogenic activity is partly regulated by phosphorylation on EPIYA motifs. Our aim was to identify effects of H. pylori infection and CagA on DDR, investigating the transcriptome of AGS cells, infected with wild-type, ΔCagA and EPIYA-phosphorylation-defective strains. Upon RNA-Seq-based transcriptomic analysis, we observed that a notable number of DDR genes were found deregulated during the infection, potentially resulting to base excision repair and mismatch repair compromise and an intricate deregulation of nucleotide excision repair, homologous recombination and non-homologous end-joining. Transcriptome observations were further investigated on the protein expression level, utilizing infections of AGS and GES-1 cells. We observed that CagA contributed to the downregulation of Nth Like DNA Glycosylase 1 (NTHL1), MutY DNA Glycosylase (MUTYH), Flap Structure-Specific Endonuclease 1 (FEN1), RAD51 Recombinase, DNA Polymerase Delta Catalytic Subunit (POLD1), and DNA Ligase 1 (LIG1) and, contrary to transcriptome results, Apurinic/Apyrimidinic Endodeoxyribonuclease 1 (APE1) upregulation. Our study accentuates the role of CagA as a significant contributor of H. pylori infection-mediated DDR modulation, potentially disrupting the balance between DNA damage and repair, thus favoring genomic instability and carcinogenesis.

Factors predisposing to thrombosis after major joint arthroplasty.

BACKGROUND: Total joint arthroplasty is one of the most common options for end stage osteoarthritis of major joints. However, we must take into account that thrombosis after hip/knee arthroplasty may be related to mutations in genes encoding for blood coagulation factors and immune reactions to anticoagulants [heparin-induced thrombocytopenia (HIT)/thrombosis]. Identifying and characterizing genetic risk should help to develop diagnostic strategies or modify anticoagulant options in the search for etiological mechanisms that cause thrombophilia following major orthopedic surgery. AIM: To evaluate the impact of patients' coagulation profiles and to study specific pharmacologic factors in the development of post-arthroplasty thrombosis. METHODS: In 212 (51 male and 161 female) patients that underwent primary total hip arthroplasty (100) or total knee arthroplasty (112) due to osteoarthritis during a period of 1 year, platelet counts and anti-platelet factor 4 (PF4)/heparin antibodies were evaluated pre/postoperatively, and antithrombin III, methylenetetrahydrofolate reductase, factor V and prothrombin gene mutations were evaluated preoperatively. In a minimum follow-up of 3 years, 196 patients receiving either low-molecular-weight heparins (173) or fondaparinux (23) were monitored for the development of thrombocytopenia, anti-PF4/heparin antibodies, HIT, and thrombosis. RESULTS: Of 196 patients, 32 developed thrombocytopenia (nonsignificant correlation between anticoagulant type and thrombocytopenia, P = 0134.) and 18 developed anti-PF4/heparin antibodies (12/173 for low-molecular-weight heparins and 6/23 for fondaparinux; significant correlation between anticoagulant type and appearance of antibodies, P = 0.005). Odds of antibody emergence: 8.2% greater in patients receiving fondaparinux than low-molecular-weight heparins. Gene mutations in factor II or V (two heterozygotes for both factor V and II) were identified in 15 of 196 patients. Abnormal low protein C and/or S levels were found in 3 of 196 (1.5%) patients, while all patients had normal levels of von Willebrand factor, lupus anticoagulant, and antithrombin III. Four patients developed HIT (insignificant correlation between thrombocytopenia and antibodies) and five developed thrombosis (two had positive antibodies and two were heterozygotes for both factor II & V mutations). Thrombosis was not significantly correlated to platelet counts or HIT. The correlation of thrombosis to antibodies, factor II, factor V was P = 0.076, P = 0.043, P = 0.013, respectively. CONCLUSION: Screening of coagulation profile, instead of platelet monitoring, is probably the safest way to minimize the risk of post-arthroplasty thrombosis. In addition, fondaparinux can lead to the formation of anti-PF4/heparin antibodies or HIT.

DNA methylation profiles in chronic lymphocytic leukemia patients treated with chemoimmunotherapy.

BACKGROUND: In order to gain insight into the contribution of DNA methylation to disease progression of chronic lymphocytic leukemia (CLL), using 450K Illumina arrays, we determined the DNA methylation profiles in paired pre-treatment/relapse samples from 34 CLL patients treated with chemoimmunotherapy, mostly (n = 31) with the fludarabine-cyclophosphamide-rituximab (FCR) regimen. RESULTS: The extent of identified changes in CLL cells versus memory B cells from healthy donors was termed "epigenetic burden" (EB) whereas the number of changes between the pre-treatment versus the relapse sample was termed "relapse changes" (RC). Significant (p < 0.05) associations were identified between (i) high EB and short time-to-first-treatment (TTFT); and, (ii) few RCs and short time-to-relapse. Both the EB and the RC clustered in specific genomic regions and chromatin states, including regulatory regions containing binding sites of transcription factors implicated in B cell and CLL biology. CONCLUSIONS: Overall, we show that DNA methylation in CLL follows different dynamics in response to chemoimmunotherapy. These epigenetic alterations were linked with specific clinical and biological features.

PALB2 c.2257C>T truncating variant is a Greek founder and is associated with high breast cancer risk.

PALB2 loss-of-function variants play an important role in breast, pancreatic and possibly, ovarian and gastric cancer susceptibility. Their frequency can be influenced by founder effects, already described in some populations. Herein, we have assessed the possible founder effect of PALB2 c.2257C>T (p.Arg753*) truncating variant among Greek breast cancer patients, while investigating possible correlations with cancer diagnoses. Following a lead deriving from a background study of highly selected Greek breast cancer patients, a total of 2496 breast and 697 ovarian cancer patients were directly genotyped for the PALB2 c.2257C>T truncating variant. Consequently, haplotype analysis was conducted on identified carriers, using seven microsatellite markers. The prevalence of the PALB2 variant was 0.24% (6/2496) and 0.14% (1/697) among breast and ovarian cases, respectively. Family history seems to be an important factor for the variant identification, although not reaching statistical significance. Microsatellite analysis on 12 carriers revealed a common shared haplotype, spanning a chromosomal region of ~1.2 Mb; the variant was possibly introduced in the Greek population ~1600 years ago. The variant confers high breast cancer risk, as illustrated by comparison with publicly available control groups. Genetic testing for PALB2, especially for the Greek founder c.2257C>T truncating variant, should be seriously considered in Greek breast cancer cases, since such findings could assist appropriate clinical management for the patients and their families.

Impact of renin-angiotensin-aldosterone system polymorphisms on myocardial perfusion: Correlations with myocardial single photon emission computed tomography-derived parameters.

BACKGROUND: Renin-angiotensin-aldosterone system (RAAS) has an important role in atherosclerosis. We investigated the effects of six RAAS gene polymorphisms on myocardial perfusion. METHODS AND RESULTS: We examined 810 patients with known or suspected coronary artery disease (CAD) using stress-rest myocardial single-photon emission computed tomography. Summed stress score (SSS), summed rest score (SRS), summed difference score (SDS), transient ischemic dilation (TID), and lung/heart ratio (LHR) were recorded. The following gene polymorphisms were investigated: angiotensin-converting enzyme (ACE) insertion/deletion (I/D), angiotensinogen (AGT) M235T and T174M, angiotensin II type 1 receptor (AT1R) A1166C, renin (REN) C5312T, and angiotensin II type 2 receptor (AT2R) C3123A. The heterozygotes or homozygotes on ACE D allele were 7.54 times more likely to have abnormal SSS, while the AGT (T174M) heterozygotes were 5.19 times more likely to have abnormal SSS. The homozygotes of ACE D had significantly higher values on TID and LHR, while the AGT (T174M) heterozygotes had higher values on TID. The AT1R heterozygotes had greater odds for having SSS ≥ 3. The patients carried AT1R homozygosity of C allele had significantly higher values on TID, while heterozygotes of AT1R had significantly higher values on LHR. CONCLUSIONS: Among the polymorphisms investigated, ACE D allele had the strongest association with abnormal myocardial perfusion.

Thumb duplication: molecular analysis of different clinical types.

PURPOSE: Molecular analysis of different types of thumb duplication and identification of new suspected gene mutations. MATERIALS AND METHODS: In a series of patients operated for polydactyly, DNA was extracted from blood samples collected preoperatively. Among these, the samples of two patients with thumb duplication (Wassel types III and IV) were initially selected for molecular analysis. The method of Clinical Exome Solution was used for the study of the phenotype-involved genes. Next-generation sequencing (NGS) was performed on a NextSeq-500 Platform (Illumina), and Sophia DDM® SaaS algorithms were used for the bioinformatics analysis of the data. RESULTS: In total, 8-including 4 new-mutations were detected in CEP290 (1 mutation), RPGRIP1 (2 mutations), TMEM216 (2 mutations), FBN1 (1 mutation), CEP164 (1 mutation), and MEGF8 (1 mutation) genes. NGS revealed 3 mutated genes in the patient with Wassel III thumb duplication and 5 mutated genes in the patient with Wassel IV duplication. The molecular analysis revealed that the patients had 2 mutated genes in common, but they only shared one common mutation. CONCLUSION: The new detected mutations are most probably associated with thumb duplication, as they belong to genes with already described mutations causing ciliopathies, often including polydactyly in their phenotype. Recognition of these mutations will be helpful to prenatal diagnosis, operative treatment strategy prediction, and possible future experimental applications in gene therapy.

Contribution of RAD51D germline mutations in breast and ovarian cancer in Greece.

RAD51D gene's protein product is known to be involved in the DNA repair mechanism by homologous recombination. RAD51D germline mutations have been recently associated with ovarian and breast cancer (OC and BC, respectively) predisposition. Our aim was to evaluate the frequency of hereditary RAD51D mutations in Greek patients. To address this, we have screened for RAD51D germline mutations 609 BRCA1- and BRCA2-negative patients diagnosed with OC, unselected for age or family history, and 569 BC patients diagnosed under 55 years and with an additional relative with BC or OC. We identified four pathogenic mutations in four unrelated individuals with family history of BC and/or OC. Three of the RAD51D carriers had developed BC, while the other one was an OC patient, thus accounting for a mutation frequency of 0.16% in the OC cohort and 0.53% in the BC cohort. One of the detected mutations is novel (c.738 + 1G > A), whereas the rest had been detected previously (p.Gln151Ter, p.Arg186Ter, and p.Arg300Ter). It is noteworthy that the 4 carrier families had 13 BC cases and only 4 OC cases. Our data support that RAD51D should be implemented into the comprehensive multigene panel, as mutation carriers may benefit from the administration of PARP inhibitors.

Characterization and prevalence of two novel CHEK2 large deletions in Greek breast cancer patients.

Germline CHEK2 mutations confer increased cancer risk, for breast and other types, which is variable depending on the specific mutation. Of these, Large Genomic Rearrangements (LGRs) have been rarely reported; to date only eight LGRs have been published with just the Czech founder mutation, the deletion of exons 9 and 10, being molecularly characterized and studied extensively. The present study aimed to molecularly define and determine the contribution of two rare, apparently novel CHEK2 LGRs, among Greek breast cancer patients. These specifically involve a ~6 kb in-frame deletion of exons 2 & 3 that removes CHEK2's FHA domain and a ~7.5 kb in-frame deletion of exon 6, which removes an α-helix of CHEK2's kinase domain. The latter was identified in 5 out of 2355 (0.22%) patients tested, while haplotype analysis revealed a common disease-associated haplotype, suggesting a single common ancestor and a Greek founder. Although in-frame, this LGR is predicted to be damaging by a yeast-based functional assay and structure-function predictions. The present study highlights the existence of rare, population-specific, genomic events in a known breast cancer predisposing gene, which can explain a proportion of hereditary breast cancer. Identification of such mutation carriers is rather important since appropriate clinical actionability will be inferred.

Genetic Analysis of Syndromic and Nonsyndromic Patients With Craniosynostosis Identifies Novel Mutations in the TWIST1 and EFNB1 Genes.

INTRODUCTION: Craniosynostosis, the premature fusion of cranial sutures, is usually divided into 2 major categories: syndromic and nonsyndromic. Mutations in the FGFR1, FGFR2, FGFR3, TWIST1, and EFNB1 genes cause the common craniosynostosis syndromes Muenke, Crouzon and Crouzon with acanthosis nigricans, Apert, Pfeiffer, Saethre-Chotzen, and Craniofrontonasal. Overlapping features among craniosynostosis syndromes, phenotypic heterogeneity even within the same syndrome, especially in the case of Muenke syndrome, and inadequate clinical evaluation can lead to misdiagnosis, which molecular testing can help clarify. OBJECTIVE: The aim of this study is to investigate the underlying genetic cause in 46 patients with syndromic or nonsyndromic craniosynostosis by direct sequencing and/or microdeletion/microduplication analysis of the FGFR1-3, TWIST1, and EFNB1 genes. RESULTS: Genetic analysis identified 3 novel mutations, c.413T>C - p.(Leu138Pro) [p.(L138P)] in TWIST1, the previously reported c.373G>A - p.(Glu125Lys) [p.(E125K)], and c.717dupA - p.(Leu240IlefsTer79) [p.(L240fs)] mutation in EFNB1 gene as well as 6 previously known mutations and a heterozygous TWIST1 gene deletion. The 2 novel mutations within EFNB1 gene arose de novo, but the novel mutation p.(L138P) within TWIST1 gene was inherited from the patient's father, who was found to be mosaic for the mutation. To our knowledge, this is the first case of mosaicism described for TWIST1 gene. CONCLUSIONS: The contribution of molecular genetic analysis to the diagnosis of patients with syndromic craniosynostosis was useful because some were originally misdiagnosed. Conversely, thorough clinical evaluation can guide molecular testing and result in a correct diagnosis.

Tumor Protein 53 Gene Mutations Without 17p13 Deletion Have No Significant Clinical Implications in Chronic Lymphocytic Leukemia. Detection of a New Mutation.

BACKGROUND: The tumor protein p53 (TP53) gene may be inactivated through 17p13 deletion, somatic mutations, or both. In chronic lymphocytic leukemia (CLL) although 17p13 deletion is correlated with poor prognosis, the role of sole TP53 mutations remains controversial. MATERIALS AND METHODS: We carried out a mutation analysis of TP53 gene in 72 patients with CLL. RESULTS: Seventy-one (98.6%) patients carried the polymorphic site c.215C>G, p.Pro72Arg, but its presence was not correlated with overall survival (OS). Moreover, 19 (26.4%) patients carried a mutation of TP53. Among the eight detected mutations, to our knowledge, one (c.587G>A) has never been reported in the past. There was a correlation of the mutation burden with the stage of the disease (p=0.022), but not with OS. None of the detected mutations was individually correlated with OS. CONCLUSION: The clinical significance of TP53 mutations is still a matter of debate and larger studies and meta-analyses are required to reach an unequivocal conclusion.

Data on eNOS T786 and G894T polymorphisms and peripheral blood eNOS mRNA levels in Sickle Cell Disease.

In this article, we present data on endothelial Nitric Oxide Synthase (eNOS) gene T786C and G894T polymorphisms in Greek steady-state Sickle Cell Disease patients in comparison to healthy controls. Moreover, eNOS mRNA levels were determined in peripheral blood samples from 18 patients and 9 controls. This article complements our recently published article named "Prognostic value of eNOS T786C and G894T polymorphisms in Sickle Cell Disease" (I. Armenis, V. Kalotychou, R. Tzanetea, Z. Kontogeorgiou, D. Anastasopoulou, M. Mantzourani, M. Samarkos, K. Pantos, K. Konstantopoulos, I. Rombos, 2016) [1].