Compound Heterozygous Patients Research Articles

The spectrum of factor XI deficiency in Southeast China: four recurrent variants can explain most of the deficiencies

BackgroundFactor XI (FXI) deficiency is an autosomal hemorrhagic disorder characterized by reduced plasma FXI levels. Multiple ancestral variants in the F11 gene have been identified in Ashkenazi Jews and other selected European populations. However, there are few reports of predominant variants in Chinese and/or East Asian populations. The aim of this study is to characterize the genotypes and phenotypes of FXI deficiency and identify the predominant variants.ResultsOf the 41 FXI-deficient patients, 39 exhibited severe FXI defects, considerably more than those with partial defects. The APTT levels showed a negative correlation with FXI activity levels (coefficient=-0.584, P < .001). Only nine patients experienced mild bleeding, including one partially defective patient and eight severely defective patients. The majority of patients were referred for preoperative screenings (n = 22) and checkups (n = 14). Genetic analysis revealed that 90% of the patients had genetic defects, with 2, 16, and 19 cases of heterozygous, homozygous, and compound heterozygous patients, respectively. Seventeen variants were detected in the F11 gene (6 novel), including eleven missense variants, four nonsense variants, and two small deletions scattered throughout the F11. Of the 11 missense variants, six have not yet been studied for in vitro expression. Protein modeling analyses indicated that all of these variants disrupted local structural stability by altering side-chain orientation and hydrogen bonds. Nine variants, consisting of three missense and six null variants, were detected with a frequency of two or more. The highest allele frequency was observed in p.Q281* (21.25%), p.W246* (17.50%), p.Y369* (12.50%), and p.L442Cfs*8 (12.50%). The former two were variants specific to East Asia, while the remaining two were southeast China-specific variants.ConclusionOur population-based cohort demonstrated that no correlation between the level of FXI activity and the bleeding severity in FXI deficiency. Additionally, the prevalence of FXI deficiency may have been underestimated. The nonsense p.Q281* was the most common variant in southeast China, suggesting a possible founder effect.

The contribution and therapeutic implications of IGHMBP2 mutations on IGHMBP2 biochemical activity and ABT1 association

Mutations within immunoglobulin mu DNA binding protein (IGHMBP2), an RNA-DNA helicase, result in SMA with respiratory distress type I (SMARD1) and Charcot Marie Tooth type 2S (CMT2S). The underlying biochemical mechanism of IGHMBP2 is unknown as well as the functional significance of IGHMBP2 mutations in disease severity. Here we report the biochemical mechanisms of IGHMBP2 disease-causing mutations D565N and H924Y, and their potential impact on therapeutic strategies. The IGHMBP2-D565N mutation has been identified in SMARD1 patients, while the IGHMBP2-H924Y mutation has been identified in CMT2S patients. For the first time, we demonstrate a correlation between the altered IGHMBP2 biochemical activity associated with the D565N and H924Y mutations and disease severity and pathology in patients and our Ighmbp2 mouse models. We show that IGHMBP2 mutations that alter the association with activator of basal transcription (ABT1) impact the ATPase and helicase activities of IGHMBP2 and the association with the 47S pre-rRNA 5′ external transcribed spacer. We demonstrate that the D565N mutation impairs IGHMBP2 ATPase and helicase activities consistent with disease pathology. The H924Y mutation alters IGHMBP2 activity to a lesser extent while maintaining association with ABT1. In the context of the compound heterozygous patient, we demonstrate that the total biochemical activity associated with IGHMBP2-D565N and IGHMBP2-H924Y proteins is improved over IGHMBP2-D565N alone. Importantly, we demonstrate that the efficacy of therapeutic applications may vary based on the underlying IGHMBP2 mutations and the relative biochemical activity of the mutant IGHMBP2 protein.

Calcium, Phosphorus, and Vitamin D Levels in a Series of Cystic Fibrosis Patients: A Cross-Sectional Study.

Cystic fibrosis (CF) is a monogenic disease with different types of mutations that mainly affect the respiratory-digestive system. Calcium (Ca), phosphorus (P), and vitamin D (Vit-D) are essential nutrients for maintaining adequate growth and development, as well as key components in crucial metabolic pathways. Proper diagnosis, treatment, and response are decisive components of precision medicine. Therefore, we conducted a cross-sectional study to evaluate Ca, P, and Vit-D levels along with health and nutritional indicators, regarding their non-skeletal functions, in a series of CF patients. Anthropometric and clinical evaluation, biochemical analysis, dietary survey, and respiratory and pancreatic status were performed. Even though the results showed that all patients had normal dietary and serum Ca levels, 47% of patients had deficient Vit-D intake, 53% of patients had hypovitaminosis D, 35% had insufficient Vit-D levels, 18% had hypophosphatemia, 76% had elevated alkaline phosphate levels, 29% had hypercalciuria, and 65% had hyperphosphaturia. There were no significant differences between homozygous and compound heterozygous patients. Ca, P, and Vit-D levels were associated with body mass index; body composition; physical activity; diet; growth hormones; and the immune, liver, and kidney systems. We suggest a periodically evaluation of Ca and P losses.

What Do We Know about Neonatal Diabetes Caused by PDX1 Mutations?

Neonatal diabetes mellitus (NDM) is characterized by severe hyperglycemia, usually diagnosed in the first few months of an individual's life. It is a genetic disease and one of the main forms of monogenic diabetes. Changes in different genes have already been associated with NDM, including changes in the gene PDX1. In this review, we intend to summarize all neonatal diabetes cases caused by PDX1 mutations reported in the literature. For this purpose, we searched keywords in the literature from PubMed and articles cited by the HGMD database. The search retrieved 84 articles, of which 41 had their full text accessed. After applying the study exclusion criteria, nine articles were included. Of those articles, we detected thirteen cases of NDM associated with changes in PDX1; the majority in homozygous or compound heterozygous patients. Until now, variants in the PDX1 gene have been a rare cause of NDM; however, few studies have included the screening of this gene in the investigation of neonatal diabetes. In this review, we reinforce the importance of the PDX1 gene inclusion in genetic NGS panels for molecular diagnosis of NDM, and systematic morphological and functional exams of the pancreas when NDM is present.

IPSC-Derived Endothelial Cells Reveal LDLR Dysfunction and Dysregulated Gene Expression Profiles in Familial Hypercholesterolemia.

Defects in the low-density lipoprotein receptor (LDLR) are associated with familial hypercholesterolemia (FH), manifested by atherosclerosis and cardiovascular disease. LDLR deficiency in hepatocytes leads to elevated blood cholesterol levels, which damage vascular cells, especially endothelial cells, through oxidative stress and inflammation. However, the distinctions between endothelial cells from individuals with normal and defective LDLR are not yet fully understood. In this study, we obtained and examined endothelial derivatives of induced pluripotent stem cells (iPSCs) generated previously from conditionally healthy donors and compound heterozygous FH patients carrying pathogenic LDLR alleles. In normal iPSC-derived endothelial cells (iPSC-ECs), we detected the LDLR protein predominantly in its mature form, whereas iPSC-ECs from FH patients have reduced levels of mature LDLR and show abolished low-density lipoprotein uptake. RNA-seq of mutant LDLR iPSC-ECs revealed a unique transcriptome profile with downregulated genes related to monocarboxylic acid transport, exocytosis, and cell adhesion, whereas upregulated signaling pathways were involved in cell secretion and leukocyte activation. Overall, these findings suggest that LDLR defects increase the susceptibility of endothelial cells to inflammation and oxidative stress. In combination with elevated extrinsic cholesterol levels, this may result in accelerated endothelial dysfunction, contributing to early progression of atherosclerosis and other cardiovascular pathologies associated with FH.

Evaluation of clinical, laboratory, and molecular genetic features of patients with biotinidase deficiency.

Biotinidase deficiency (BD) is an autosomal recessive inherited metabolic disorder which results from the inability of biotin-dependent carboxylase enzymes to function due to the release and absorption of biotin, leading to neurological and cutaneous findings. In the present study, evaluation of demographic characteristics, clinical findings, laboratory results, molecular genetic characteristics, and genotype-phenotype correlations of cases with BD. Two hundred forty-seven cases were included in the study who were admitted to the Department of Pediatric Metabolism of Ankara Bilkent City Hospital after being identified with potential BD through the Newborn Screening Program (NBS), during family screening or based on suspicious clinical findings, or following the detection of a pathogenic variant in a BTD genetic analysis during the period of October 2020 and February 2022. The medical files of the cases were reviewed retrospectively. An analysis of the admission routes of all cases to our clinic revealed 89.5% NBS, 5.7% family screening, and 4.9% suspicious clinical findings suggestive of BD. Complete enzyme deficiency was identified in 19.8%, partial enzyme deficiency in 55.1%, and heterogenous enzyme deficiency in 9.7%. The most common pathogenic variants were c.1270G > C (p.Asp424His), c.410G > A (p.Arg137His), and c.38_44delGCGCTGinsTCC (p.Cys13Phefs*36) in BTD gene. The c.1270G > C variant was most common in patients with cutaneous symptoms. The c.410G > A and c.38_44delGCGCTGinsTCC variants were more common in the patients with neurological symptoms. The mean activity level in patients with the c.1270G > C homozygous variant was statistically significantly higher than the mean activity level in the c.1270G > C compound heterozygous patients and the activity level of patients without the c.1270G > C variant. The mean activity level in c.410G > A homozygous patients was statistically significantly lower than the mean activity level of the c.410G > A compound heterozygous patients and the activity level of patients without the c.410G > A variant. In the course of our study, four new pathogenic variants were detected, namely: c.190G > A (p.Glu64Lys), c.249 + 5G > T, c.228delA (p.Val77*), and c.682A > G (p.Ile228Val). Conclusions: The present study has determined the clinical and genetic spectrum of a large group of patients with BD in a single center. The frequent mutations in our study were similar to those reported in literature, and four novel variants were also described. What is Known: • Biotinidase deficiency is an autosomal recessive, treatable inborn error of metabolism. Two hundred ninety-four pathogenic variants in the BTD gene have been identified and the c.1270G > C variant is the most frequent BTD gene mutation in both Turkey and around the world. What is New: • Four new pathogenic variants (c.190G > A, p.Glu64Lys; c.249 + 5G > T; c.228delA, p.Val77*; and c.682A > G, p.Ile228Val) have been identified. It is believed that the c.38_44delGCGGCTGinsTCC variant is more commonly seen in individuals with ocular issues; however, further genotype-phenotype correlations are needed.

Expression analysis of NF1-mutated alleles in a rare compound heterozygous spinal NF1 patient by digital PCR.

Neurofibromatosis type 1 (NF1) is a heterogeneous neurocutaneous disorder. Spinal neurofibromatosis (SNF) is a distinct clinical entity of NF1, characterized by bilateral neurofibromas involving all spinal nerve roots. Although both forms are caused by intragenic heterozygous variants of NF1, missense variants have been associated with SNF, according to a dominant inheritance model causing haploinsufficiency. Most patients carry pathogenic variants in one of theNF1 alleles; nevertheless, patients with both NF1-mutated copies have been described. Interestingly, all NF1 variants carried by the known SNF compound heterozygotes were missense/splicing variants or in-frame insertion-deletions. To investigate whether there is a differential expression of NF1 variant alleles in an NF1 compound heterozygous SNF patient possibly contributing to clinical phenotype. We performed an allele-specific expression study, by chip-based digital PCR, in an SNF family carrying two NF1 missense variants. We evaluated the expression levels of the two NF1-mutated alleles both carried by the compound heterozygous SNF patient and his relatives. Both alleles were expressed at comparable levels in the patient and hyper-expressed compared to the wild-type alleles of healthy controls. Here we provide new insights into expression studies of NF1-mutated transcripts suggesting that a novel pathogenetic mechanism, caused by gain-of-function variants, could be associated with SNF. Further studies should be performed in larger cohorts, opening new perspectives in the NF1 pathogenesis comprehension.

When a synonymous mutation breaks the silence in a thalassaemia patient.

Synonymous mutations were considered to lack functional roles in human diseases; however, distinguishing deleterious synonymous mutations from benign ones is still a challenge. In this article, we identified a deleterious synonymous mutation β-codon 16 (C>T). HBB: c.51C>T, in compound heterozygous form with known β-thalassaemia mutation patients who clinically presented as non-transfusion-dependent thalassaemia (NTDT). A total of 9 families with 11 compound heterozygous index cases were reported. In the heterozygous state, codon 16 (C>T) mutation results in borderline HbA2 (3.18 ± 0.36%) and slightly reduced RBC indices (RBCs: 4.73 ± 0.75 × 106 /μL, Hb: 12.26 ± 2.60 g/dL, MCV: 79.48 ± 8.40 fL, MCH: 25.95 ± 4.15 pg). The compound heterozygous patients showed elevated HbA2 (5.98 ± 1.17%) and HbF (12.75 ± 7.51%) and presented clinically as NTDT with a mean Hb of 6.95 ± 1.29 g/dL. Many of them were dependent on few transfusions and had mild splenomegaly. Of the 11 patients, 5 (45.4%) were treated with hydroxyurea. This study highlights the clinical significance of synonymous mutation, when inherited with other β-thalassaemia mutations leading to the phenotype of NTDT. Thus, the study would help to improve screening protocols for β-thalassaemia carriers, which will ultimately improve the prevention programme.

Novel pathological variants of NHP2 affect N-terminal domain flexibility, protein stability, H/ACA RNP complex formation and telomerase activity.

Telomere biology disorders (TBDs) are characterized by short telomeres, premature aging, bone marrow failure, and cancer predisposition. Germline mutations in NHP2, encoding for one component of the telomerase cofactor H/ACA RNA binding complex together with Dyskerin, NOP10, and GAR1, have been previously reported in rare cases of TBDs. Here we report two novel NHP2 variants (NHP2-A39T and NHP2-T44M) identified in a compound heterozygous patient affected by premature aging, bone marrow failure/myelodysplastic syndrome, and gastric cancer. Although still able to support cell viability, both variants reduce the levels of hTR, the telomerase RNA component, and telomerase activity, expanding the panel of NHP2 pathological variants. Furthermore, both variants fail to be incorporated in the H/ACA RNA binding complex when in competition with wild-type endogenous NHP2, and the lack of incorporation causes their drastic proteasomal degradation. By RoseTTAFold prediction followed by molecular dynamics simulations, we reveal a dramatic distortion of residues 33-41, which normally position on top of the NHP2 core, as the main defect of NHP2-A39T. Furthermore, we predicted high flexibility and the misplacement of the N-terminal region (residues 1-24) in NHP2-T44M and, to a lower degree, in NHP2-A39T. Since deletion of amino acids 2-24 cause a reduction in NHP2 levels only in the presence of wild type NHP2, while deletion of amino acids 2-38 completely disrupts NHP2 stability, we propose that the two variants are mis-incorporated into the H/ACA binding complex due to the altered dynamics of the first 23 amino acids and/or the distortion of the residues 25-41 loop.

Allelic Frequency of DPYD Genetic Variants in Patients With Cancer in Spain: The PhotoDPYD Study.

Identifying polymorphisms in the dihydropyrimidine dehydrogenase (DPYD) gene is gaining importance to be able to predict fluoropyrimidine-associated toxicity. The aim of this project was to describe the frequency of the DPYD variants DPYD*2A (rs3918290); c.1679T>G (rs55886062); c.2846A>T (rs67376798) and c.1129-5923C>G (rs75017182; HapB3) in the Spanish oncological patients. Cross-sectional and multicentric study (PhotoDPYD study) conducted in hospitals located in Spain designed to register the frequency of the most relevant DPYD genetic variants in oncological patients. All oncological patients with DPYD genotype were recruited in the participant hospitals. The measures determined where the presence or not of the 4 DPYD previously described variants. Blood samples from 8054 patients with cancer from 40 different hospitals were used to determine the prevalence of the 4 variants located in the DPYD gene. The frequency of carriers of one defective DPYD variant was 4.9%. The most frequently identified variant was c.1129-5923C>G (rs75017182) (HapB3), in 2.9%, followed by c.2846A>T (rs67376798) in 1.4%, c.1905 + 1G>A (rs3918290, DPYD*2A) in 0.7% and c.1679T>G (rs55886062) in 0.2% of the patients. Only 7 patients (0.08%) were carrying the c.1129-5923C>G (rs75017182) (HapB3) variant, 3 (0.04%) the c.1905 + 1G>A (rs3918290, DPYD*2A) and one (0.01%) the DPYD c.2846A>T (rs67376798, p.D949V) variant in homozygosis. Moreover, 0.07% were compound heterozygous patients, 3 carrying the DPYD variants DPYD*2A + c.2846A>T, 2 the DPYD c.1129-5923C>G + c.2846A>T and one the DPYD*2A + c.1129-5923C>G variants. Our results demonstrate the relatively high frequency of DPYD genetic variants in the Spanish patient with cancer population, which highlights the relevance of their determination before initiating a fluoropirimidine-containing regimen.

Clinical spectrum and genotype-phenotype associations in Finnish patients with Wilson's disease

Genotype-phenotype correlation data covering all ages of Wilson's disease onset in Caucasian patients are limited. We therefore analyzed genotype-phenotype correlations in a retrospective cohort of Finnish patients. Six homozygous (HoZ) and 11 compound heterozygous (CoHZ) patients were included. There were no differences in the presence/absence of hepatic, neurological, psychiatric or any symptoms at diagnosis (p > 0.30 for all) between HoZ and CoHZ patients, but HoZ patients had an earlier age of diagnosis (median 6.7 versus 34.5; p = 0.003). Severe liver affliction was almost exclusively associated with the p.H1069Q variant. Patients with p.H1069Q had a later mean age of diagnosis (30.2 ± 11.6 vs. 8.7 ± 4.9 years; p < 0.001) compared to those without. There were no differences in the presence/absence of hepatic, neurological, psychiatric or any symptoms at diagnosis between p.H1069Q-positive and p.H1069Q-negative patients (p > 0.54 for all). These results suggest that population-specific factors may partly explain the high clinical variability of Wilson's disease.

Distinct effects of two hearing loss–associated mutations in the sarcomeric myosin MYH7b

For decades, sarcomeric myosin heavy chain proteins were assumed to be restricted to striated muscle where they function as molecular motors that contract muscle. However, MYH7b, an evolutionarily ancient member of this myosin family, has been detected in mammalian nonmuscle tissues, and mutations in MYH7b are linked to hereditary hearing loss in compound heterozygous patients. These mutations are the first associated with hearing loss rather than a muscle pathology, and because there are no homologous mutations in other myosin isoforms, their functional effects were unknown. We generated recombinant human MYH7b harboring the D515N or R1651Q hearing loss-associated mutation and studied their effects on motor activity and structural and assembly properties, respectively. The D515N mutation had no effect on steady-state actin-activated ATPase rate or load-dependent detachment kinetics but increased actin sliding velocity because of an increased displacement during the myosin working stroke. Furthermore, we found that the D515N mutation caused an increase in the proportion of myosin heads that occupy the disordered-relaxed state, meaning more myosin heads are available to interact with actin. Although we found no impact of the R1651Q mutation on myosin rod secondary structure or solubility, we observed a striking aggregation phenotype when this mutation was introduced into nonmuscle cells. Our results suggest that each mutation independently affects MYH7b function and structure. Together, these results provide the foundation for further study of a role for MYH7b outside the sarcomere.

The most common European HINT1 neuropathy variant phenotype and its case studies.

HINT1 is an ubiquitous homodimeric purine phosphoramidase belonging to the histidine-triad superfamily. In neurons, HINT1 stabilizes the interaction of different receptors and regulates the effects of their signaling disturbances. Changes in HINT1 gene are associated with autosomal recessive axonal neuropathy with neuromyotonia. Aim of the study was detailed description of patients' phenotype with HINT1 homozygous NM_005340.7: c.110G>C (p.Arg37Pro) variant. Seven homozygous and three compound heterozygous patients were recruited and evaluated using standardized tests for CMT patients, in four patients' nerve ultrasonography was performed. The median age of symptom onset was 10 years (range 1-20), with initial complaints being distal lower limb weakness with gait impairment, combined with muscle stiffness, more pronounced in the hands than in the legs and worsened by cold. Arm muscles became involved later, presenting with distal weakness and hypotrophy. Neuromyotonia was present in all reported patients and is thus a diagnostic hallmark. Electrophysiological studies demonstrated axonal polyneuropathy. Impaired mental performance was observed in six out of ten cases. In all patients with HINT1 neuropathy, ultrasound examination showed significantly reduced muscle volume as well as spontaneous fasciculations and fibrillations. The nerve cross-sectional areas of the median and ulnar nerves were closer to the lower limits of the normal values. None of the investigated nerves had structural changes. Our findings broaden the phenotype of HINT1-neuropathy and have implications for diagnostics and ultrasonographic evaluation of HINT1-neuropathy patients.

Changes in cystic fibrosis transmembrane conductance regulator protein expression prior to and during elexacaftor-tezacaftor-ivacaftor therapy.

Background: Defects in expression, maturation or function of the epithelial membrane glycoprotein CFTR are causative for the progressive disease cystic fibrosis. Recently, molecular therapeutics that improve CFTR maturation and functional defects have been approved. We aimed to verify whether we could detect an improvement of CFTR protein expression and maturation by triple therapy with elexacaftor-tezacaftor-ivacaftor (ELX/TEZ/IVA). Methods: Rectal suction biopsies of 21 p.Phe508del homozygous or compound heterozygous CF patients obtained pre- and during treatment with ELX/TEZ/IVA were analyzed by CFTR Western blot that was optimized to distinguish CFTR glycoisoforms. Findings: CFTR western immunoblot analysis revealed that-compared to baseline-the levels of CFTR protein increased by at least twofold in eight out of 12 patients upon treatment with ELX/TEZ/IVA compared to baseline (p < 0.02). However, polydispersity of the mutant CFTR protein was lower than that of the fully glycosylated wild type CFTR Golgi isoform, indicating an incompletely glycosylated p.Phe508el CFTR protein isoform C* in patients with CF which persists after ELX/TEZ/IVA treatment. Interpretation: Treatment with ELX/TEZ/IVA increased protein expression by facilitating the posttranslational processing of mutant CFTR but apparently did not succeed in generating the polydisperse spectrum of N-linked oligosaccharides that is characteristic for the wild type CFTR band C glycoisoform. Our results caution that the lower amounts or immature glycosylation of the C* glycoisoform observed in patients' biomaterial might not translate to fully restored function of mutant CFTR necessary for long-term provision of clinical benefit.

Mutations in the non-coding RNU4ATAC gene affect the homeostasis and function of the Integrator complex.

Various genetic diseases associated with microcephaly and developmental defects are due to pathogenic variants in the U4atac small nuclear RNA (snRNA), a component of the minor spliceosome essential for the removal of U12-type introns from eukaryotic mRNAs. While it has been shown that a few RNU4ATAC mutations result in impaired binding of essential protein components, the molecular defects of the vast majority of variants are still unknown. Here, we used lymphoblastoid cells derived from RNU4ATAC compound heterozygous (g.108_126del;g.111G>A) twin patients with MOPD1 phenotypes to analyze the molecular consequences of the mutations on small nuclear ribonucleoproteins (snRNPs)formation and on splicing. We found that the U4atac108_126del mutant is unstable and that the U4atac111G>A mutant as well as the minor di- and tri-snRNPs are present at reduced levels. Our results also reveal the existence of 3'-extended snRNA transcripts in patients' cells. Moreover, we show that the mutant cells have alterations in splicing of INTS7 and INTS10 minor introns, contain lower levels of the INTS7 and INTS10 proteins and display changes in the assembly of Integrator subunits. Altogether, our results show that compound heterozygous g.108_126del;g.111G>A mutations induce splicing defects and affect the homeostasis and function of the Integrator complex.

Genetic Study of Early Onset Parkinson’s Disease in Cyprus

Parkinson's Disease (PD) is a multifactorial neurodegenerative disease characterized by motor and non-motor symptoms. The etiology of PD remains unclear. However, several studies have demonstrated the interplay of genetic, epigenetic, and environmental factors in PD. Early-onset PD (EOPD) is a subgroup of PD diagnosed between the ages of 21 and 50. Population genetic studies have demonstrated great genetic variability amongst EOPD patients. Hence, this study aimed to obtain a genetic landscape of EOPD in the Cypriot population. Greek-Cypriot EOPD patients (n = 48) were screened for variants in the six most common EOPD-associated genes (PINK1, PRKN, FBXO7, SNCA, PLA2G6, and DJ-1). This included DNA sequencing and Multiplex ligation-dependent probe amplification (MLPA). One previously described frameshift variant in PINK1 (NM_032409.3:c.889del) was detected in five patients (10.4%)-the largest number to be detected to date. Copy number variations in the PRKN gene were identified in one homozygous and 3 compound heterozygous patients (8.3%). To date, the pathogenic variants identified in this study have explained the PD phenotype for 18.8% of the EOPD cases. The results of this study may contribute to the genetic screening of EOPD in Cyprus.

Lysosomal alterations and decreased electrophysiological activity in CLN3 disease patient-derived cortical neurons.

CLN3 disease is a lysosomal storage disorder associated with fatal neurodegeneration that is caused by mutations in CLN3, with most affected individuals carrying at least one allele with a 966 bp deletion. Using CRISPR/Cas9, we corrected the 966 bp deletion mutation in human induced pluripotent stem cells (iPSCs) of a compound heterozygous patient (CLN3 Δ 966 bp and E295K). We differentiated these isogenic iPSCs, and iPSCs from an unrelated healthy control donor, to neurons and identified disease-related changes relating to protein synthesis, trafficking and degradation, and in neuronal activity, which were not apparent in CLN3-corrected or healthy control neurons. CLN3 neurons showed numerous membrane-bound vacuoles containing diverse storage material and hyperglycosylation of the lysosomal LAMP1 protein. Proteomic analysis showed increase in lysosomal-related proteins and many ribosomal subunit proteins in CLN3 neurons, accompanied by downregulation of proteins related to axon guidance and endocytosis. CLN3 neurons also had lower electrophysical activity as recorded using microelectrode arrays. These data implicate inter-related pathways in protein homeostasis and neurite arborization as contributing to CLN3 disease, and which could be potential targets for therapy.

Genotype-Phenotype Study of β-Thalassemia Patients in Sabah

β-thalassemia is a serious public health problem in Sabah due to its high prevalence. This study aimed to investigate the effects of different types of β-globin gene mutations, coinheritance with α-globin gene mutations, XmnI-Gγ, and rs368698783 polymorphisms on the β-thalassemia phenotypes in Sabahan patients. A total of 111 patients were included in this study. The sociodemographic profile of the patients was collected using a semi-structured questionnaire, while clinical data were obtained from their medical records. Gap-PCR, ARMS-PCR, RFLP-PCR, and multiplex PCR were performed to detect β- and α-globin gene mutations, as well as XmnI-Gγ and rs368698783 polymorphisms. Our data show that the high prevalence of β-thalassemia in Sabah is not due to consanguineous marriages (5.4%). A total of six different β-globin gene mutations were detected, with Filipino β°-deletion being the most dominant (87.4%). There were 77.5% homozygous β-thalassemia patients, 16.2% compound heterozygous β-thalassemia patients, and 6.3% β-thalassemia/Hb E patients. Further evaluation on compound heterozygous β-thalassemia and β-thalassemia/Hb E patients found no concomitant α-globin gene mutations and the rs368698783 polymorphism. Furthermore, the XmnI-Gγ (−/+) genotype did not demonstrate a strong impact on the disease phenotype, as only two of five patients in the compound heterozygous β-thalassemia group and two of three patients in the β-thalassemia/Hb E group had a moderate phenotype. Our findings indicate that the severity of the β-thalassemia phenotypes is closely related to the type of β-globin gene mutations but not to the XmnI-Gγ and rs368698783 polymorphisms.

Compound Heterozygosis in AADC Deficiency and Its Complex Phenotype in Terms of AADC Protein Population.

Aromatic amino acid decarboxylase (AADC) deficiency is a rare monogenic disease due to mutations in the ddc gene producing AADC, a homodimeric pyridoxal 5′-phosphate-dependent enzyme. The disorder is often fatal in the first decade and is characterized by profound motor impairments and developmental delay. In the last two years, there has been a net rise in the number of patients and variants identified, maybe also pushed by the ongoing gene therapy trials. The majority of the identified genotypes are compound heterozygous (about 70%). Efforts are underway to reach early diagnosis, find possible new markers/new fast methods, and predict clinical outcome. However, no clear correlation of genotype-to-phenotype exists to date. Nevertheless, for homozygous patients, reliable results have been obtained using genetic methods combined with available computational tools on crystal structures corroborated by biochemical investigations on recombinant homodimeric AADC variants that have been obtained and characterized in solution. For these variants, the molecular basis for the defect has been suggested and validated, since it correlates quite well with mildness/severity of the homozygous phenotype. Instead, prediction for compound heterozygous patients is more difficult since complementation effects could happen. Here, by analyzing the existing literature on compound heterozygosity in AADC deficiency and other genetic disorders, we highlight that, in order to assess pathogenicity, the measurement of activity of the AADC heterodimeric variant should be integrated by bioinformatic, structural, and functional data on the whole protein constellation theoretically present in such patients. A wider discussion on symptomatic heterozygosity in AADC deficiency is also presented.

Novel Germline TET2 Mutations in Two Unrelated Patients with Autoimmune Lymphoproliferative Syndrome-Like Phenotype and Hematologic Malignancy.

Somatic mutations in the ten-eleven translocation methylcytosine dioxygenase 2 gene (TET2) have been associated to hematologic malignancies. More recently, biallelic, and monoallelic germline mutations conferring susceptibility to lymphoid and myeloid cancer have been described. We report two unrelated autoimmune lymphoproliferative syndrome-like patients who presented with T-cell lymphoma associated with novel germline biallelic or monoallelic mutations in the TET2 gene. Both patients presented a history of chronic lymphoproliferation with lymphadenopathies and splenomegaly, cytopenias, and immune dysregulation. We identified the first compound heterozygous patient for TET2 mutations (P1) and the first ALPS-like patient with a monoallelic TET2 mutation (P2). P1 had the most severe form of autosomal recessive disease due to TET2 loss of function resulting in absent TET2 expression and profound increase in DNA methylation. Additionally, the immunophenotype showed some alterations in innate and adaptive immune system as inverted myeloid/plasmacytoid dendritic cells ratio, elevated terminally differentiated effector memory CD8 + T-cells re-expressing CD45RA, regulatory T-cells, and Th2 circulating follicular T-cells. Double-negative T-cells, vitamin B12, and IL-10 were elevated according to the ALPS-like suspicion. Interestingly, the healthy P1's brother carried a TET2 mutation and presented some markers of immune dysregulation. P2 showed elevated vitamin B12, hypergammaglobulinemia, and decreased HDL levels. Therefore, novel molecular defects in TET2 confirm and expand both clinical and immunological phenotype, contributing to a better knowledge of the bridge between cancer and immunity.