Coexistence of Genetic Diseases Is a New Clinical Challenge: Three Unrelated Cases of Dual Diagnosis.

Technological advancements in molecular genetics and cytogenetics have led to the diagnostic definition of complex or atypical clinical pictures. In this paper, a genetic analysis identifies multimorbidities, one due to either a copy number variant or a chromosome aneuploidy, and a second due to biallelic sequence variants in a gene associated with an autosomal recessive disorder. We diagnosed the simultaneous presence of these conditions, which co-occurred by chance, in three unrelated patients: a 10q11.22q11.23 microduplication and a homozygous variant, c.3470A>G (p.Tyr1157Cys), in the WDR19 gene associated with autosomal recessive ciliopathy; down syndrome and two variants, c.850G>A; p.(Gly284Arg) and c.5374G>T; p.(Glu1792*), in the LAMA2 gene associated with merosin-deficient congenital muscular dystrophy type 1A (MDC1A); and a de novo 16p11.2 microdeletion syndrome and homozygous variant, c.2828G>A (p.Arg943Gln), in the ABCA4 gene associated with Stargardt disease 1 (STGD1). The possibility of being affected by two relatively common or rare inherited genetic conditions would be suspected when signs and symptoms are incoherent with the primary diagnosis. All this could have important implications for improving genetic counseling, determining the correct prognosis, and, consequently, organizing the best long-term follow-up.

A Systematic Review and Meta-Analysis of the Association between the FV H1299R Variant and the Risk of Recurrent Pregnancy Loss.

This study evaluated the association between the H1299R factor V (FV) variant (rs1800595) and recurrent pregnancy loss (RPL). Pubmed (MEDLINE) and Embase (OVID) bibliographic databases were searched from the inception to 31 May 2022 to identify suitable articles according to PRISMA and MOOSE guidelines. We included observational studies, case-control studies, cross-sectional studies, and cohort studies reporting a numerical and well-distinguished Het or Hom status of the H1299R variant obtained through PCR or other biochemical techniques and comparing RPL patients with a healthy control group. The review protocol was registered at PROSPERO (CRD42022330077). Two authors independently screened studies, extracted data, and carried out the risk of bias assessment using the Newcastle Ottawa scale (NOS). A meta-analysis was performed with RevMan software Version 5.4 using an odds ratio (OR) with an M-H, random effect, and 95% CI. We included 13 clinical studies for a total of 1669 RPL patients and 1466 healthy women as a control group. H1299R variant was slightly associated with RPL albeit without significance (OR 1.18, 95% CI: 0.78-1.80, p = 0.44). Subgroup analyses considering H1299R in heterozygosity (OR 1.13, 95% CI: 0.76-1.67, p = 0.56) and in homozygosity (OR: 2.11, 95% CI: 0.74-6.01, p = 0.16) revealed a similar trend. Lastly, we evaluated the association between H1299R and RPL based on the number of previous miscarriages (≥2 or ≥3). This comprehensive systematic review and meta-analysis sheds light on the specific influence of the H1299R variant in the F5 gene on RPL, constituting valid support for medical care during pregnancy and genetic counseling.

H1299R Variant in Factor V and Recurrent Pregnancy Loss: A Systematic Review and Meta-Analysis Protocol.

Recurrent pregnancy loss (RPL) is defined as the loss of two or more pregnancies, affecting approximately 1 to 3% of women worldwide. Scientific data highlight a possible correlation between thrombophilic genetic variants and RPL. H1299R variant in the factor V gene would lead to an increased thrombotic risk associated with frequent miscarriages. However, the data are often conflicting, making this an interesting question for further investigations by evaluating genotype-phenotype correlations to improve the clinical management and genetic counseling of couples. A systematic review and meta-analysis will follow the preferred reporting elements for systematic review and meta-analysis protocols (PRISMA-P). The Pubmed (MEDLINE) and Embase (OVID) databases will be explored to identify suitable articles based on inclusion and exclusion criteria. Inclusion criteria are: (a) H1299R genotyping with clear data reported, referred to as Heterozygous (Het) and/or Homozygous (Hom); (b) articles written in English; (c) analyses of only RPL female patients having at least two or more previous pregnancy losses and compared with a control group. This analysis will present selected scientific evidence, addressing the questions concerning the association between the H1299R variant and RPL, hoping to clarify this still unresolved issue. PROSPERO registration number: CRD42022330077.

12q21 Interstitial Deletions: Seven New Syndromic Cases Detected by Array-CGH and Review of the Literature.

Interstitial deletions of the long arm of chromosome 12 are rare, with a dozen patients carrying a deletion in 12q21 being reported. Recently a critical region (CR) has been delimited and could be responsible for the more commonly described clinical features, such as developmental delay/intellectual disability, congenital genitourinary and brain malformations. Other, less frequent, clinical signs do not seem to be correlated to the proposed CR. We present seven new patients harboring non-recurrent deletions ranging from 1 to 18.5 Mb differentially scattered across 12q21. Alongside more common clinical signs, some patients have rarer features such as heart defects, hearing loss, hypotonia and dysmorphisms. The correlation of haploinsufficiency of genes outside the CR to specific signs contributes to our knowledge of the effect of the deletion of this gene-poor region of chromosome 12q. This work underlines the still important role of copy number variations in the diagnostic setting of syndromic patients and the positive reflection on management and family genetic counseling.

CNVs inform the biological network of Autism spectrum disorder.

Autism spectrum disorder (ASD) is a heterogeneous condition linked to an anomalous neurodevelopment. Although the underlying causes of ASD are not well described, literature data strongly suggests a genetic component, with a complex inheritance pattern. It has recently been observed that CNVs (copy number variation) may play an important role in ASD manifestation and partially explain the complex heritability of this tract. Another factor That adds another level of complexity to ASD is its potential genetic heterogeneity. In this paper, we hypothesize that the different patterns of alteration within individuals with ASD may converge towards the same function. We genotyped a sample of 107 individuals through aCGH analysis for CNVs that were related (by localization) to approximately 1400 genes. The genes were tested for functional interactions and clustered in functional groups. We highlighted a functional genetic cluster of 256 genes potentially related to ASD. These altered genes may contribute to the same function, alterations of which increase the risk of ASD. After testing our functional cluster for biological functions, processes related to oxidative stress, immune system and energy metabolism are the pathways potentially involved with the biological alterations underlying ASD.

Molecular Pathways within Autism Spectrum Disorder Endophenotypes.

Autism spectrum disorder (ASD) is a condition that includes a number of neurodevelopmental mental disorders. Recent genetic/genomic investigations have reported an increased prevalence of copy number variations (CNVs) in individuals with autism. Despite the extensive evidence of a genetic component, the genes involved are not known and the background is heterogeneous among subjects. As such, it is highly likely that multiple events (molecular cascades) are implicated in the development of autism. The aim of this work was to shed some light on the biological background behind this condition. We hypothesized that the heterogeneous alterations found within different individuals may converge into one or more specific biological functions (pathways) linked to the heterogeneous phenotypes commonly observed in subjects with ASD. We analyzed a sample of 107 individuals for CNV alterations and checked the genes located within the altered loci (1366). Then, we characterized the subjects for distinct phenotypes. After creating subsamples based on symptoms, the CNVs related to each specific symptom were used to create distinct networks associated with each phenotype (18 in total in the sample under analysis). These networks were independently clustered and enriched to identify potential common pathways involved in autism and variably combined with the clinical phenotype. The first 10 pathways of the analysis are discussed.

High mobility group box 1 and markers of oxidative stress in human cord blood.

BACKGROUND: Parturition induces considerable oxidative stress and many inflammatory mediators, such as high mobility group box 1 (HMGB1), are involved from the beginning of the pregnancy to birth. The aim of the present study was to evaluate serum cord blood concentration of diacron-reactive oxygen metabolites (d-ROM), biological antioxidant potential (BAP), and HMGB1 to investigate the perinatal oxidative status of neonates and correlation with mode of delivery, as well as the influence of labor. METHODS: The subjects consisted of 214 neonates delivered at University Hospital "G. Martino", Messina, in a 6 months period. Venous blood samples were collected from the umbilical cord after cord separation. RESULTS: Umbilical cord venous blood HMGB1 was significantly higher in the spontaneous vaginal delivery (SVD) group than in the elective or emergency cesarean section (CS) group (P = 0.018). Regarding labor, there was no significant difference in HMGB1 concentration in umbilical vein blood between the spontaneous and induced labor groups (P = 0.250). Furthermore, d-ROM was significantly different between the SVD group and the elective or emergency CS group (P = 0.044). BAP concentration, however, was not significantly different, not even with regard to mode of labor. CONCLUSION: Oxidation is higher in newborns delivered by SVD than in those delivered by CS, and HMGB1 may be involved in the mechanisms of birth, and responsible for decidual modifications that lead to birth.

Genetic analysis of the human insulin-like 3 gene in pediatric patients with testicular torsion.

PURPOSE: Testicular torsion (TT) mainly affects boys under 18 years old. To avoid orchiectomy, TT requires an immediate operative management. The etiology of TT is still controversial. Observed familiar recurrence suggests the presence of a genetic involvement. The INSL3 gene consists of two exons, and it is specifically expressed in fetal and adult Leydig cells. In transgenic mice, deletion of this gene was observed an increased testicular mobility and testicular torsion. We have hypothesized the possible involvement of the INSL3 gene as a predisposing factor of human TT. METHODS: We performed genetic analysis in 25 pediatric patients with unilateral and intravaginal TT (left, n = 13, 56%; right, n = 12, 48%). The age of the patients ranged from 1 to 16 years (median age n = 10.4 ± 5.46 years). In this study, we included two first male cousins affected by TT. Venous peripheral blood samples was obtained after parental written informed consent. RESULTS: The Thr60Ala polymorphism was detected in exon 1 of INSL3 gene and other 2 rarer variants (rs1047233 and rs1003887) were identified in the 3' untranslated region. These variants are prevalent in patients with TT instead of healthy subjects. CONCLUSIONS: Additional studies in a larger population are needed to better understand the clinical consequence of the INSL 3 variations founded. This would allow in the future to identify the patients at risk of TT to improve clinical management.

FTL c.-168G>C Mutation in Hereditary Hyperferritinemia Cataract Syndrome: A New Italian Family.

We describe a new Italian family with 7 members affected by hereditary hyperferritinemia cataract syndrome (HHCS), an uncommon autosomal dominant disease caused by mutations of the iron-responsive element (IRE) of the ferritin light chain (FTL) gene determining its overexpression. The family diagnosis of HHCS took place after finding high ferritin levels in a 6-year-old girl. Seven members of the family had bilateral and symmetrical cataracts, normal iron, and hematological parameters except for high serum ferritin levels. About 160 families/unrelated cases with HHCS are known worldwide. This report documents a second Italian family, with a c.-168G>C mutation that is located in the highly conserved 3-nucleotide bulge structure of the FTL in the 5' untranslated region. This case shows how important the family history is in reaching a correct diagnosis and avoiding unnecessary and invasive analysis. HHCS should be considered in the differential diagnosis of childhood hyperferritinemia, especially in the presence of normal transferrin saturation.

A child with severe iron-deficiency anemia and a complex TMPRSS6 genotype.

OBJECTIVES: We report a case of a 7-year-old girl with severe hypochromic microcytic anemia, who was unresponsive to classical iron supplements. We suspected IRIDA, iron-refractory iron-deficiency anemia, a genetic iron metabolism disorder, caused by TMPRSS6 variations. TMPRSS6 encodes matriptase-2, a negative regulator of hepcidin, and its pathological variants are related to normal to high levels of hepcidin. We analyzed the TMPRSS6 gene and we improved clinical management of the patient, selecting the appropriate supplementation therapy. Intervention & Technique: The parenteral iron therapy was started, but the patient was only partially responsive and the anemia persisted. To confirm the diagnosis, the TMPRSS6 gene sequence was analyzed by DNA sequencing and other relevant biochemical parameters were evaluated. RESULTS: The TMPRSS6 sequence analysis showed a complex genotype with a rare heterozygous missense variant, in addition to other common polymorphisms. The serum hepcidin value was normal. We unexpectedly observed a normalization of patient's hemoglobin (Hb) levels only after liposomal iron treatment. DISCUSSION AND CONCLUSION: The proband was symptomatic for IRIDA during a critical phase of growth and development, but we did not find a clearly causative genotype. A long-term result, improving stably patient's Hb levels, was obtained only after liposomal iron supplementation. Children may be at greater risk for iron deficiency and the degree of anemia as well as the response to the iron supplements varies markedly patient to patient. Here, we show the importance of comprehensive study of these patients in order to collect useful information about genotype-phenotype association of genes involved in iron metabolism.

Genotoxic effect of iron overload and disease complications in transfused ß thalassaemic patients.

In previously reported studies, we observed significantly high genotoxicity biomarkers in regularly transfused thalassaemic patients, thus, in this study, we better investigated the genotoxic effect of iron overload and of thalassaemia complications, including their drug treatments. The assessment was performed in 64 regularly transfused thalassaemic patients using cytokinesis-block micronucleus and comet assays. All patients were splenectomised and undergoing iron chelation therapy. To reduce hypoxia-induced oxidative damage, the patients with haemoglobin levels <9.5 g/dL were excluded. Serum concentrations of ferritin, iron, transferrin and the percentage of transferrin saturation, as well as cardiac and hepatic T2* magnetic resonance imaging, were considered to evaluate serum and organ siderosis.All genotoxic biomarkers significantly differed between patients and healthy subjects. Iron intake via blood transfusions was inversely related to percentage of DNA in tail. The disease complications affecting endpoints were active Hepatitis C virus infection, drug therapy for osteoporosis (i.e. bisphosphonates) and hormone replacement therapy for hypogonadism.The results, highlighting the combined effect of iron overload and, mainly, disease complications, including their respective pharmacological treatments, confirmed the increased cancer risk in thalassaemic patients.

Soluble hemojuvelin in transfused and untransfused thalassaemic subjects.

OBJECTIVE: The hemojuvelin-bone morphogenetic protein axis is the principal iron-dependent mechanism of hepcidin regulation. The determination of soluble hemojuvelin (sHJV) levels could allow for a better understanding of the pathophysiological mechanisms of hepcidin regulation in thalassaemia. METHOD: We have assessed sHJV in 45 transfused and 15 untransfused thalassaemic patients in comparison with 15 healthy subjects, evaluating its relationships with some parameters of iron overload, anaemia and erythropoiesis. RESULTS: Untransfused thalassaemic patients had more severe anaemia and erythropoietic activity, while in transfused patients, the transfused RBCs reduced % reticulocytes and sTfR, increased serum indices of iron overload and iron stores in the liver (low MRI T2* values). sHJV levels were higher in patients than in controls and in untransfused in comparison with transfused patients. In the transfused group, we also found that sHJV values are significantly related to serum ferritin, cardiac MRI T2* and growth differentiation factor 15 and are sensitive to hepatitis C virus infection. CONCLUSION: These results suggest that sHJV synthesis seems to be affected by an erythropoietic/hypoxic signal in untransfused patients that have severe anaemia, while in regularly transfused subjects, it is influenced by iron stores.

The role of anaemia in oxidative and genotoxic damage in transfused ß-thalassaemic patients.

OBJECTIVES: Redox imbalance and genotoxic damage are commonly observed in ß thalassaemic patients. The aim of this study was to assess the role of anaemia in oxidative and genotoxic damage in regularly transfused thalassaemic patients, undergoing iron chelation therapy. METHODS: We studied the relationships of haematological, biochemical and clinical parameters with oxidative (reactive oxygen species and 8-oxo-7,8-dihydro-2'-deoxyguanosine) and genotoxic biomarkers (Comet assay and cytokinesis-block micronucleus test) in blood samples from 105 patients. To reduce the early effect of redox-active iron, samples were collected when pharmacokinetics of the iron chelators ensured their maximum effectiveness. The transfusion regimen, cardiac and hepatic magnetic resonance imaging T2* were evaluated to characterize the patient cohort. Labile plasma iron (LPI) was also assayed. RESULTS: Haemoglobin level had a significant effect on ROS, %DNA in the tail and micronuclei-micronucleated cell frequency (p < 0.05). Higher Hb values reduced redox imbalance. LPI, detectable in 50.5% of patients, was related to the number of apoptotic and necrotic lymphocytes (p = 0.03), demonstrating the cytotoxic effect of iron. DISCUSSION: The results highlight that an adequate transfusion regimen is essential to limit oxidative and genotoxic damage in ß-thalassemic patients undergoing chelation therapy. CONCLUSION: Owing to the higher risk of cancer in the thalassaemic cohorts, specific genotoxicity/oxidative biomarkers should be monitored in order to ameliorate and formulate more personalized disease management.

Effects of strontium ranelate on bone mass and bone turnover in women with thalassemia major-related osteoporosis.

Subjects affected by thalassemia major (TM) often have reduced bone mass and increased fracture risk. Strontium ranelate (SrR) is an effective treatment for postmenopausal and male osteoporosis. To date, no data exist on the use of SrR in the treatment of TM-related osteoporosis. Our aim was to evaluate the effects of SrR on bone mineral density (BMD), bone turnover markers and inhibitors of Wnt signaling (sclerostin and DKK-1). Twenty-four TM osteoporotic women were randomized to receive daily SrR 2 g or placebo in addition to calcium carbonate (1,000 mg) and vitamin D (800 IU). BMD at the lumbar spine and femoral neck, bone turnover markers (C-terminal telopeptide of procollagen type I [CTX], bone-specific alkaline phosphatase [BSAP]) and insulin-like growth factor-1 (IGF-1), sclerostin and DKK-1 were assessed at baseline and after 24 months. Back pain was measured by visual analog scale (VAS) every 6 months. After 24 months, TM women treated with SrR had increased their spine BMD values in comparison to baseline (p < 0.05). Moreover, they also exhibited a reduction of CTX and sclerostin levels (but not DKK-1) and exhibited an increase of BSAP and IGF-1 (p < 0.05); however, no significant changes were observed in the placebo group. In the SrR group, a reduction of back pain was observed after 18 months in comparison to baseline (p < 0.05) and after 24 months in comparison to placebo (p < 0.05). Our study reports for the first time the effects of SrR in the treatment of TM-related osteoporosis. SrR treatment improved BMD and normalized bone turnover markers, as well as lowering sclerostin serum levels.

Oxidative damage and genotoxicity biomarkers in transfused and untransfused thalassemic subjects.

Chronic anemia and tissue hypoxia increase intestinal iron absorption and mitochondrial impairment in thalassemic patients. Regular blood transfusions improve hemoglobin levels but determine an iron overload that induces reactive oxygen species (ROS) overproduction. The aim of this study was to assess cellular oxidative damage by detection of ROS, lipid peroxidation, 8-oxo-dG, and mitochondrial transmembrane potential (Δψ(m)) in transfused and untransfused thalassemic patients. We have also evaluated genotoxicity by CBMN and comet assay. Our data show that ROS and lipid hydroperoxides are significantly higher in thalassemic patients than in controls, especially in untransfused thalassemia intermedia patients. Moreover, the latter have a significant decrease in Δψ(m) that highlights the energetic failure in hypoxic state and the ROS overproduction in the respiratory chain. 8-OHdG levels are higher in thalassemics than in controls, but do not differ significantly between the two patient groups. Both genotoxicity biomarkers highlight the mutagenic potential of hydroxyl radicals released by iron in the Fenton reaction. Values for percentage of DNA in the comet tail and micronuclei frequency, significantly higher in transfused patients, could also be due to active hepatitis C virus infection and to the many drug treatments. Our biomonitoring study confirms the oxidative damage in patients with thalassemia major and shows an unexpected cellular oxidative damage in untransfused thalassemic patients. In addition to iron overload, the results highlight the important role played by hypoxia-driven mitochondrial ROS overproduction in determining oxidative damage in ß-thalassemias.

Role of genetic pattern on bone mineral density in thalassemic patients.

OBJECTIVES: To evaluate the role of genetic background in osteoporosis/osteopenia development in beta-Thalassemia Major patients. DESIGN AND METHODS: The influence of VDR (FokI, BsmI) as well as COLIA1 (Sp1) gene polymorphisms on BMD was investigated in 40 patients. RESULTS: Although the examined gene polymorphisms did not significantly affect BMD variations in our population, BsmI was found to display beneficial effects on patient response to alendronate therapy. CONCLUSION: Genetic factors retain a potential role for improvement of osteoporosis management in thalassemic patients.

Long-term treatment with deferiprone in a L1 veteran.

We studied a patient with mild beta-thalassaemia major under treatment with the oral chelator deferiprone (DFP or L1) for about 10 yr (L1 veteran). Due to poor compliance with desferrioxamine, the patient started compassionate use of DFP at an age of 23 yr with a serum ferritin of 5200 microg/L. Monitoring iron overload by SQUID biosusceptometry revealed a dramatic decrease of liver iron concentrations from 4500 to 950 microg/g(liver) within 9.5 yr. A good clinical response to chelation treatment with DFP was observed together with an improvement of liver and cardiac function and a reduction in the hepatitis virus load.