Outcome of non-visualization of fetal gallbladder on second-trimester ultrasound: cohort study and systematic review of literature.

OBJECTIVES: To investigate the ultrasound characteristics and outcome of fetuses with non-visualization of the fetal gallbladder (NVFGB) followed in our tertiary university hospital, and to provide a comprehensive review of the literature on prenatal findings and outcome of NVFGB. METHODS: NVFGB was defined as non-visualization of the gallbladder on two targeted ultrasound examinations performed within a 1-week period. First, we reviewed the medical records of NVFGB cases managed in our center over a 9-year period. Then, we performed a systematic review of the literature to identify studies on NVFGB. The incidence of chromosomal anomalies, later visualization of the gallbladder, gallbladder agenesis, cystic fibrosis and biliary atresia was assessed in fetuses with isolated and non-isolated NVFGB. The role of hepatic enzyme measurements in the diagnosis of cystic fibrosis and biliary atresia in fetuses with NVFGB was also reviewed. RESULTS: Sixteen cases of NVFGB were followed in our center, in 10 (62.5%) of which it was an isolated finding. The incidence of biliary atresia was 12.5% and that of gallbladder agenesis was 12.5%, while no case of cystic fibrosis was reported. The gallbladder was visualized later in pregnancy or postnatally in 43.8% and 25.0% of cases, respectively. A total of seven studies, including our cohort, involving a total of 280 NVFGB cases, met the inclusion criteria for the systematic review. Overall, 20.5% of fetuses had an associated ultrasound anomaly, and the incidence of chromosomal anomaly in this group was 20.4%. In cases with isolated NVFGB, the incidence of chromosomal anomaly was 1.9%. In fetuses with normal karyotype and isolated NVFGB, the gallbladder was later visualized in 70.4% of cases, while the incidence of gallbladder agenesis, cystic fibrosis and biliary atresia was 25.2%, 3.1% and 4.8%, respectively. In fetuses with non-isolated NVFGB, the incidence of cystic fibrosis and biliary atresia was 23.1% and 18.2%, respectively. The negative predictive value of amniotic fluid enzyme levels for the prediction of severe disease (including biliary atresia or cystic fibrosis) ranged between 94% and 100% when evaluated before 22 weeks' gestation, and dropped to 88% after 22 weeks. CONCLUSIONS: In cases with persistent NVFGB, the risk of a severe postnatal condition should be considered. A detailed ultrasound scan should be offered and parents tested for cystic fibrosis gene mutation. An invasive procedure for karyotyping and measurement of liver enzyme concentrations before 22 weeks constitutes a reasonable work-up. Copyright © 2019 ISUOG. Published by John Wiley & Sons Ltd.

No visualización de la vesícula biliar fetal en la ecografía del segundo trimestre del embarazo: estudio de cohortes y revisión sistemática de la literatura sobre el resultado postnatal OBJETIVOS: Investigar las características ecográficas y los resultados de los fetos con no visualización de la vesícula biliar fetal (NVFGB, por sus siglas en inglés) a los que se ha dado seguimiento en un hospital universitario terciario, y ofrecer una revisión exhaustiva de la literatura sobre los hallazgos prenatales y los resultados de la NVFGB. MÉTODOS: La NVFGB se definió como la no visualización de la vesícula biliar en dos exámenes ecográficos específicos realizados en un período de una semana. Primero, se revisó los registros médicos de los casos de NVFGB tratados en este hospital durante un período de 9 años. Luego, se realizó una revisión sistemática de la literatura para identificar estudios sobre NVFGB. Se evaluó la incidencia de anomalías cromosómicas, la visualización posterior de la vesícula biliar, la agenesia vesicular, la fibrosis quística y la atresia biliar en fetos con NVFGB aislada y no aislada. También se examinó la función de las mediciones de las enzimas hepáticas en el diagnóstico de la fibrosis quística y la atresia biliar en fetos con NVFGB. RESULTADOS: Se siguieron dieciséis casos de NVFGB en este centro hospitalario, lo cual fue un hallazgo aislado en 10 de ellos (62,5%). La incidencia de atresia biliar fue del 12,5% y la de agenesia vesicular del 12,5%, mientras que no se reportó ningún caso de fibrosis quística. La vesícula biliar se visualizó más tarde en el embarazo o después del parto en el 43,8% y 25,0% de los casos, respectivamente. Un total de siete estudios cumplieron los criterios de inclusión para la revisión sistemática, incluidos los de la cohorte del mencionado hospital, con un total de 280 casos de NVFGB. En total, el 20,5% de los fetos presentaban una anomalía ecográfica asociada, y la incidencia de anomalías cromosómicas en este grupo fue del 20,4%. En los casos con NVFGB aislada, la incidencia de anomalías cromosómicas fue del 1,9%. En los fetos con cariotipo normal y NVFGB aislada, la vesícula biliar se visualizó posteriormente en el 70,4% de los casos, mientras que la incidencia de agenesia vesicular, fibrosis quística y atresia biliar fue del 25,2%, 3,1% y 4,8%, respectivamente. En los fetos con NVFGB no aislada, la incidencia de fibrosis quística y atresia biliar fue del 23,1% y 18,2%, respectivamente. El valor predictivo negativo de los niveles de enzimas del líquido amniótico para la predicción de una enfermedad grave (como la atresia biliar o la fibrosis quística) se situó entre el 94% y el 100% cuando se evaluó antes de las 22 semanas de gestación, y bajó al 88% después de las 22 semanas. CONCLUSIONES: En casos con NVFGB persistente, se debe considerar el riesgo de una condición postnatal severa. Se debe ofrecer una ecografía detallada y la madre y el padre deben someterse a pruebas para detectar mutaciones del gen de la fibrosis quística. Un examen diagnóstico razonable incluye un procedimiento invasivo para el cariotipado y la medición de las concentraciones de enzimas hepáticas antes de las 22 semanas.

Lymphocyte subset reconstitution after HLA-identical placental blood transplantation (PBT) or PBT plus bone marrow transplantation (BMT) in three children with beta-thalassemia major.

The kinetics of circulating lymphoid cells were evaluated in three children suffering from beta-thalassemia major after HLA-identical sibling placental blood transplant (PBT) in one patient and placental blood plus bone marrow transplantation (BMT) in two patients. Recovery of the main lymphocyte subsets, as determined by phenotype analysis of circulating PBMCs, was complete within 2 months after transplant. NK (CD56+) cells were the first to appear in peripheral blood, followed by T (CD3+, CD2+, CD7+) and B (CD19+) cells. Of the T lymphocytes, the CD8+ were the first to reconstitute, but recovery of CD4+ cells was also rapid and within 6 months these T cells reached normal values. The expression of CD57 by NK or T cells was slightly delayed. The evaluation of RA and RO isoform expression of the CD45 molecule showed a prevalence of the CD45RA antigen with a ratio of 2-3:1. In the PBT only patient, T cells expressing the CD45RO antigen prevailed in the early post-transplant period. Severe or chronic GVHD was not observed. This experience demonstrates that reconstitution of lymphocyte subsets is successful in genetic hematological diseases after transplantation of HLA-identical placental blood or placental blood plus bone marrow from healthy or heterozygous siblings. Bone Marrow Transplantation (2000) 26, 743-747.

Genotype and phenotype correlation in glucose-6-phosphate dehydrogenase deficiency.

BACKGROUND AND OBJECTIVES: Glucose-6-phosphate dehydrogenase (G6PD) deficiency is the most common erythrocytic enzymatic disorder in Italy and is characterized by wide clinical, biochemical and molecular variability. We studied the clinical and hematologic data from 54 G6PD-deficient, unrelated males from the Apulia region. DESIGN AND METHODS: Analyses for enzymatic activity, G6PD electrophoresis and molecular typing were performed on all subjects. Thirty-nine subjects (72.2%) showed a severe G6PD deficiency (<10% residual enzymatic activity) and 15 subjects (27.8%) a moderate deficiency (10--60% residual activity). RESULTS: The Mediterranean variant was found in 48.2% of cases, the Seattle variant in 33.3%, the A- variant in 7.45% and the Montalbano variant in 3.7%; the variant was not identified in four subjects. Thirty-two patients (59.2%) were asymptomatic; of these, 37.04% demonstrated acute hemolytic crises induced mainly by ingestion of fava beans and 3.7% had had neonatal jaundice. Acute hemolytic anemia was found in 53.8% of subjects with the Mediterranean variant, in 5.5% with the Seattle variant, in 100% with the A-variant and 0% with the Montalbano variant. INTERPRETATION AND CONCLUSIONS: Enzymatic activity was shown to be a poor predictive parameter of acute hemolytic crises and was not correlated with clinical features. Subjects with Mediterranean or A- variants had a more severe clinical phenotype which was not related to enzymatic activity. The Seattle, and probably the Montalbano, variant appears to have a milder clinical expression.

HFE gene mutations an Apulian population: allele frequencies.

Hereditary hemochromatosis (HH) is an autosomal recessive trait regarding iron metabolism frequently found in Caucasian populations. The C282Y mutation of the HFE gene, held responsible for HH, has been identified as the major genetic basis for the phenotypic expression of HH whereas two additional mutations of the HFE H63D and S65C gene appear to be associated with a milder form of HH. A high allele frequency of C282Y and H63D has been reported in Northern European populations. In Italy, the overall allele frequency was 0.5% for the C282Y mutation, 12.6% for the H63D mutation and 1.1% for the S65C mutation. In this study, we evaluated the allele frequency of the three principal HFE mutations (C282Y, H63D, S65C) together with eight additional mutations (V53M, H63H, Q127H, E168Q, E168stop, W169stop, V59M, Q238P) in 500 healthy Apulian subjects. No subject homozygous for the C282Y mutation was found while 3% of subjects were heterozygous for this mutation. Heterozygosity and homozygosity for the H63D mutation were 26 and 1%, respectively. Only five subjects were heterozygous for the S65C mutation. Overall, the allele frequency was 1.5% for C282Y, 14% for H63D, 0.5% for S65C and 0% for the other mutations. The transferrin saturation (TS) was significantly higher in subjects heterozygous for the H63D mutations with respect to subjects with a normal genotype, though all were within the normal range. No statistically significant difference in the allele frequency was noted in the Apulian population compared to that in Northern and Southern Italy.

Analysis of HLA-DRB1*-A* and -B* alleles in prenatal diagnosis for determination of maternal contamination in fetal DNA.

During chorionic villi sampling for prenatal diagnosis with molecular biology techniques, contamination by maternal decidua frequently occurs and can lead to misinterpretation of the test results. To avoid such problems, we present a new method for appraising maternal contamination of fetal DNA, based on genomic typing of the highly variable human leukocyte antigen (HLA) locus-DRB1*, locus A* and locus B* regions by genetic amplification with sequence-specific primers and PCR. Fetal DNA samples obtained for beta-thalassemia diagnosis were analysed after artificial contamination with increasing maternal DNA concentrations ranging from 0.5 to 10% (0.5, 1, 3, 5 and 10%). The approach was found to be rapid, specific, reproducible and highly sensitive and permits recognition of 1-3% contamination by maternal DNA concentrations. The system currently used for detecting maternal DNA contamination in fetal samples is the analysis of polymorphic loci by variable number of tandem repeats and/or short tandem repeats. We propose that the analysis of HLA alleles may provide a valid alternative or complement to this system.

Prenatal diagnosis of beta-thalassaemia using fetal erythroblasts enriched from maternal blood by a novel gradient.

We have assessed a new technique for the isolation of fetal erythroblasts from maternal blood for the non-invasive prenatal diagnosis of pregnancies at risk of beta-thalassaemia. This method relies on the separation of erythroblasts from maternal nucleated cells by a novel step gradient and high speed centrifugation. In four of the six cases examined, single erythroblasts were identified by immunohistochemistry for zeta (zeta) globin. These were individually micromanipulated and analysed by single cell polymerase chain reaction (PCR) and subsequent sequencing of the region of beta-globin locus where the mutations most common to the region of Puglia, Italy, are clustered. In each of the four instances where fetal erythroblasts were identified by antibody staining, the fetal beta-globin genotype was correctly determined. To date, this represents the largest series of non-invasive prenatal diagnoses performed for this haemoglobinopathy.

Molecular basis of beta-thalassemia intermedia in a southern Italian region (Puglia).

We investigated the molecular bases for a mild phenotype by alpha-, beta- and gamma-globin gene analyses in 22 patients with transfusion-independent thalassemia intermedia (15) or a late-presenting form of thalassemia major (7) originating from Puglia, a region of southern Italy. Twenty-two patients with thalassemia major served as controls. The beta+ IVS-I nt 6 of the beta-globin gene and the C----T substitution at position -158 5' of the G gamma-globin gene were detected more frequently in patients with thalassemia intermedia or late-presenting thalassemia major considered together as compared to those affected by typical transfusion-dependent thalassemia major. Three of 15 patients with thalassemia intermedia had the triple alpha-globin gene arrangement in the heterozygous (2) or homozygous state (1) in association with heterozygous beta zero-thalassemia. From these results, we may conclude that the inheritance of a mild beta-thalassemia allele such as the beta+ IVS-I nt 6 mutation, in the homozygous or heterozygous state, the coinheritance with homozygous beta zero-thalassemia of the -158 (C----T) G gamma gene promoter mutation and the presence of heterozygous beta-thalassemia/triple alpha-globin gene arrangement are the most common reasons accounting for the development of attenuated forms of beta-thalassemia in Puglia.

Promoter mutations producing mild beta-thalassaemia in the Italian population.

In this study we have investigated the molecular basis for a mild form of beta-thalassaemia in three patients of Italian descent. In two, belonging to different families and affected by a mild and late-presenting form of thalassaemia major, direct sequencing of amplified DNA detected a C----T substitution at position -87 of the beta-globin gene in the compound heterozygous state either with codon 39 nonsense mutation or beta +IVSI, nt 110 mutation. The -87 (C----T) mutation has been previously described, in combination with the beta +IVSI, nt 110 mutation, in a single patient with thalassaemia intermedia. Both our patients showed a more severe phenotype as compared to that resulting from compound heterozygosity for a severe beta-thalassaemia mutation and another promoter mutation (-87, C----G) at the same position. In the third patient with the thalassaemia intermedia phenotype, we detected a novel promoter mutation, consisting in a C----A substitution at position -86, in combination with the codon 39 nonsense mutation. The results of this study indicate that different nucleotide substitutions affecting the proximal CACCC box of the beta-globin gene in combination with severe beta-thalassaemia, produce a mild form of thalassaemia ranging in severity from thalassaemia intermedia to late-presenting thalassaemia major.