Genotype-phenotype correlation of 17 cases of Pompe disease in Spanish patients and identification of 4 novel GAA variants.

BACKGROUND: Pompe disease (PD) is an autosomal recessive metabolic disorder caused by pathogenic variants in the acid α-glucosidase gene (GAA) that produces defects in the lysosomal acid α-1,4-glucosidase. We aimed to identify genetic variations and clinical features in Spanish subjects to establish genotype-phenotype correlation. METHODS: A total of 2637 samples of patients who showed symptoms or susceptible signs of PD were enrolled in this observational study. Enzymatic activity was detected by fluorometric techniques and the genetic study was carried out using Next-Generation Sequencing. RESULTS: Fourteen different variants from 17 diagnosed patients were identified, seven males and nine females with LOPD (mean age 36.07, SD 20.57, range 7-64) and a 2-day-old boy with IOPD, four genetic variants had not been described in the literature previously, including a homozygous variant. In all of them α-glucosidase activity was decreased. Muscle weakness, respiratory distress, exercise intolerance, hypotonia, dysphagia and myalgia were commonly observed in patients. CONCLUSIONS: This study report four new genetic variants that contribute to the pathogenic variants spectrum of the GAA gene. We confirm that patients in Spain have a characteristic profile of a European population, with c.-32-13T>G being the most prevalent variant. Furthermore, it was confirmed that the c.236_246delCCACACAGTGC pathogenic variant in homozygosity is associated with early disease and a worse prognosis.

Accuracy diagnosis improvement of Fabry disease from dried blood spots: Enzyme activity, lyso-Gb3 accumulation and GLA gene sequencing.

The purpose of this study was to examine the applicability of the use of samples in dried blood spot (DBS) for the definitive diagnosis of Fabry disease (FD) in males and females and to compare the diagnostic role of α-galactosidase A activity (α-Gal A), levels of lyso-Gb3 and sequencing of the GLA gene in screening patients with suspected FD. Measurement of α-Gal A activity in suspected FD patients in DBS was made followed by lyso-Gb3 determination and GLA gene sequencing. Of the 2381 subjects analyzed, FD was confirmed in 24 patients. Thirteen different variants were considered like pathogenic, five of which had not been previously described (c.143A > G; c.455A > C; c.487G > T; c.554delA; c.1045_1046insA). None of the patients with normal enzyme activity had FD confirmation. The DBS measurement of α-Gal A was more sensitive than lyso-Gb3 levels in both men and women. Definitive diagnosis of FD from a single DBS is possible, allowing samples to be easily sent from anywhere to the reference laboratory.

Genotype/phenotype relationship in Gaucher disease patients. Novel mutation in glucocerebrosidase gene.

Objectives Gaucher disease (GD) is the most common inherited lysosomal storage disease, caused by mutations in acid ß-glucosidase (GBA) gene. This study aimed to identify mutations in Andalusia patients with GD and their genotype-phenotype correlation. Methods Descriptive observational study. University Hospital Virgen del Rocio patients diagnosed from GD from 1999 to 2019 were included. Demographic and clinical data, ß-glucocerebrosidase activity, variants pathogenic in GBA gene and biomarkers for monitoring treatment were collected from digital medical record. Results Twenty-six patients with aged between 1 day and 52 years were studied. A total of six mutations described as pathogenic and one mutation not described above [c.937T>C (p.Tyr313His)] were identified in the GBA gene, four patients were homozygotes and 22 compound heterozygotes. Twenty-four patients were diagnosed in non-neuropathic form (type 1) and two cases presented neurological involvement (type 2 or 3). The most common variant was c.1226A>G (p.Asn409Ser), which was detected in 24 patients, followed by c.1448T>C (p.Leu483Pro) variant, identified in 13 patients. The c.1448T>C (p.Leu483Pro) mutation has been presented in the most severe phenotypes with neurological involvement associated with type 2 and 3 GD, while c.1226A>G (p.Asn409Ser) mutation has not been associated with neurological alterations. Splenomegaly and bone disease were the most frequent clinical manifestations, and thrombocytopenia was the most common hematological disorder. Conclusions The c.1226A>G (p.Asn409Ser) and c.1448T>C (p.Leu483Pro) mutations were the most common. The c.937T>C (p.Tyr313His) was identified as a novel mutation. The c.1448T>C (p.Leu483Pro) mutation was associated with neurological alterations and c.1226A>G (p.Asn409Ser) mutation has not been associated it.

Noninvasive prenatal diagnosis by cell-free DNA screening for fetomaternal HPA-1a platelet incompatibility.

BACKGROUND: The development of new noninvasive approaches for the diagnosis of human platelet antigen (HPA)-1 fetomaternal incompatibility has become of great interest. These approaches allow determination of whether the fetus is incompatible or not with the mother and a decision on antenatal therapy to avoid fetal or neonatal alloimmune thrombocytopenia (FNAIT). The objective of this work was to perform rapid, noninvasive prenatal test for HPA-1ab fetal antigen detection after the detection of an HPA-1-homozygous mother by using plasma cell-free DNA (cfDNA). STUDY DESIGN AND METHODS: The HPA-1 genotypes of 142 pregnant women and 17 nonpregnant controls were retrospectively determined by high-resolution melting (HRM) polymerase chain reaction (PCR). Coamplification at lower denaturation temperature (COLD) HRM PCR was performed to determine the fetal genotype analyzing cfDNA from all HPA-1bb pregnant women. RESULTS: After the HRM analysis, the following genotypes were identified: HPA-1aa (71.13%), HPA-1bb (2.8%), and HPA-1ab (26.06%). Four HPA-1bb-homozygous pregnant women were carrying an incompatible fetus. Plasma samples from these mothers were analyzed by HRM COLD-PCR. Homozygous HPA-1bb pregnant women carrying an HPA-1ab-heterozygous fetus did not group with either the HPA-1ab or the HPA-1bb controls. Thus, COLD-PCR analysis allows the detection of HPA-1ab-heterozygous fetuses carried by homozygous mothers during first weeks of pregnancy. CONCLUSION: The fetal genotype from HPA-1bb-homozygous women was detected by a noninvasive prenatal test as soon as 12 weeks of gestation.

Detection of p53 Mutations in Circulating DNA of Transplanted Hepatocellular Carcinoma Patients as a Biomarker of Tumor Recurrence.

p53 is the most commonly mutated gene in malignant human cancers. To detect p53 mutations in circulating DNA (cirDNA) of transplanted hepatocellular carcinoma (HCC) patients could be an interesting approach to know of any tumor recurrence. In this study, our objective was to determine the utility of this method in the diagnosis and the prognosis of HCC tumor recurrence.Twenty four liver transplanted HCC patients were included in the study together with a group of healthy controls. Detection of the specific p53 mutation in cirDNA was performed by high-resolution melting PCR (HRM-PCR) and COLD-PCR immediately before the transplantation. Serum anti-p53 was also determined using a p53-autoantibody ELISA kit.The results of the HRM-PCR and COLD-PCR showed two well-differentiated groups of transplanted patients after normalization by healthy controls. These data allow us to distinguish between patients with p53 mutated cirDNA and those with wild type cirDNA. Moreover, we have found that most of p53 mutated patients also presented elevated anti-p53 antibodies. The present results indicate that it is possible to detect mutated p53 genes with the cirDNA and that this could be used as a biomarker of tumor recurrence during the clinical evolution of the transplanted patients.

Screening of KRAS Mutation in Pre- and Post-Surgery Serum of Patients Suffering from Colon Cancer by COLD-PCR HRM.

Genomic characterization of cell-free circulating tumour DNA (ctDNA) may offer an opportunity to assess clonal dynamics throughout the course of a patient's illness. The existence of KRAS driver mutations in colon cancer patients is determinant to decide their treatment and to predict their outcome. DNA is extracted automatically from 400 µL of serum using the MagNa Pure Compact with the Nucleic Acid Isolation Kit I. DNA amplification, COLD-PCR and HRM were performed in the same run in the Light Cycler 480.We found three different situations: pre- and post-surgical samples grouped with the negative control, pre-surgical samples appear to group with the positive control and the post-surgical samples appear to group with the negative control and finally both samples, pre- and post-surgical ones, appear to be grouped with the positive control. COLD-PCR HRM is a cost-effective way for screening one of the most common driver mutations to predict the worst prognosis in colorectal cancer.

Non-invasive Prenatal Diagnosis of Feto-Maternal Platelet Incompatibility by Cold High Resolution Melting Analysis.

Fetal and Neonatal alloimmune thrombocytopenia (FNAIT) is a condition which could occur when pregnant women develop an alloimmunization against paternally inherited antigens of the fetal platelets. Approximately 80 % of FNAIT cases are caused by anti-HPA-1a, about 15 % by anti-HPA-5b and 5 % by other HPA antibodies. Only 2 % of the total population is HPA-1a negative (HPA-1b1b). The HPA-1a allele differs by one single nucleotide from HPA-1b allele, yet it represents around 27 % of total severe thrombocytopenias. HPA-1 was studied in serum cDNA from 12 volunteer pregnant women to determine their HPA-1 genotype by HRM (high resolution melting) PCR. When an homozygous HPA-1 gene was detected in a mother, a COLD HRM was performed to determine whether or not the fetal genotype differs from the mother's.The differences in the melting curve shapes allow us to accurately distinguish the three pregnants genotypes. The fetal heterozygous genotype of homozygous pregnant women was correctly detected by COLD PCR HRM in maternal serum. HPA-1 genotyping by HRM may be a useful aproach for genotyping all pregnant women in inexpensively. Moreover, when HPA-1 homozygosis was detected in a pregnant woman, fetal heterozygosis may be diagnosed by COLD HRM to select pregnancies for preventive monitoring.

Evaluation of the State of Transplanted Liver Health by Monitoring of Organ-Specific Genomic Marker in Circulating DNA from Receptor.

The evaluation of the transplanted liver health by non-invasive approaches may offer an improvement in early clinical intervention. As transplanted organs have genomes that are distinct from the host's genome, the quantification of the specific DNA of the donated liver in the patient serum will allow us to obtain information about its damage. We evaluated the state of transplanted liver health by monitoring the RH gene in serum circulating DNA (cirDNA) from 17 recipient and donor mismatched for this gene. cirDNA RH gene was quantified by RT- PCR before, at the moment of transplantation (day 0) and during the stay at the intensive care unit. Beta-globin cirDNA was quantified as a general cellular damage marker. Patients were grouped based on clinical outcomes: (A) patients with no complication; (B) patients that accepted the organ but suffered other complications; (C) patients that suffered organ rejection. All patients showed an increased cirDNA levels at day 0 that decreased until patient stabilization. Patients from groups A and B showed low levels of the RH gene cDNA during the follow-up, with an increase of beta-globin gene at the moment of any clinical complication. Patients from group C showed an increase in the RH gene during rejection.