Molecular basis of pyruvate kinase deficiency among Tunisians: description of new mutations affecting coding and noncoding regions in the PKLR gene.

INTRODUCTION: Pyruvate kinase (PK) deficiency is one of the most common hereditary nonspherocytic hemolytic anemias worldwide with clinical manifestations ranging from mild to severe hemolysis. However, investigation of this enzymopathy is lacking in Tunisia. We report here a pioneer investigation of PK deficiency among Tunisian cases referred to our laboratory for biological analysis of unknown cause of hemolytic anemia. METHODS: Two hundred and fifty-three patients with unknown cause of hemolytic anemia have been addressed to our laboratory in order to investigate for red blood cells genetic disorders. Red cell enzyme activities were measured by standard methods, and molecular analysis was performed by DNA sequencing. The interpretation of mutation effect and the molecular modeling were performed by using specific software. RESULTS: Six different PKLR mutations were found (c.966-1G>T; c.965+1G>A; c.721G>T; c.1163C>A; c.1456C>T; c.1537T>A), among which four are described for the first time. Genotype-phenotype correlations for the novel missense mutations were investigated by three-dimensional structure analysis. CONCLUSION: This study provides important data of PK deficiency among Tunisians. It might be followed by a large neonatal screening to determine the spectrum of PK mutations and identify potential deficient patients for an early medical follow-up.

The lactase -13910C>T polymorphism (rs4988235) is associated with overweight/obesity and obesity-related variables in a population sample of Portuguese young adults.

BACKGROUND/OBJECTIVES: Several studies reported associations of the lactase gene (LCT) polymorphism -13910C>T (rs4988235) with obesity-related variables and obesity in adults. This study aimed to replicate previously reported associations in a population sample of Portuguese young adults. SUBJECTS/METHODS: We genotyped 447 subjects from central and northern regions of Portugal (mean age 20.81±4.24 years) for the lactase variant -13910C>T (rs4988235), using TaqMan probes. Anthropometric variables (weight, height and body fat) were measured using standardized procedures and body mass index (BMI) (kg/m2) was calculated. RESULTS: Frequency of genotypes was 35.8% CC (lactase nonpersistent, LNP), 48.1% CT and 16.1% TT, consistent with Hardy-Weinberg equilibrium (P=1). The frequency for the minor -13910 T allele was 0.402. Assuming a dominance model for the lactase persistence (LP) minor T-allele, linear regression models showed statistically significant associations between the LP genotype CT/TT and BMI, fat mass and weight (ß=1.114, P=0.003; ß=1.309, P=0.007 and ß=2.67, P=0.021, respectively) after adjustment for age and sex. In concordance, logistic regression showed significant association between LP genotype CT/TT and overweight/obesity (OR=1.77; CI 1.08-2.92; P=0.023), as well as with high fat percentage ranges (OR=1.58; CI 1.01-2.46; P=0.041), when adjusting for age and sex. No significant interaction was obtained between the LCT polymorphism and physical activity for BMI (Pint=0.454) or FAT % (Pint=0.421). CONCLUSIONS: In the Portuguese sample of young adults, the lactase -13910C>T polymorphism revealed significant associations with the obesity-related anthropometric variables BMI, fat mass and weight, and previously observed associations with the obesity risk were also confirmed.

Detection of new pathogenic mutations in patients with congenital haemolytic anaemia using next-generation sequencing.

INTRODUCTION: Congenital haemolytic anaemia (CHA) refers to a group of genetically heterogeneous disorders, mainly caused by changes in genes encoding globin chains, cytoskeletal proteins and red cell enzymes, in which accurate diagnosis can be challenging with conventional techniques. METHODS: To set-up a comprehensive assay for detecting mutations that could improve aetiological diagnosis, we designed a custom panel for sequencing coding regions from 40 genes known to be involved in the pathogenesis of CHA, using the Ion Torrent™ (Thermo Fisher Scientific, S.L. Waltham, MA, USA) Personal Genome Machine (PGM) Sequencer. A control group of 16 samples with previously known mutations and a test group of 10 patients with unknown mutations were included for assay validation and application, respectively. RESULTS: In the test group, we identified pathogenic mutations in all cases: four patients had novel mutations in genes related to membrane defects (SPTB, ANK1, SLC4A1 and EPB41), four were homozygous or compound heterozygous for mutations in genes related to enzyme deficiencies (GPI, TPI1 and GSS), one had a mutation in the HBB gene and another presented a homozygous mutation in the ADAMTS13 gene. CONCLUSIONS: Ion PGM sequencing with our custom panel is a highly efficient way to detect mutations causing haemolytic anaemia, including new variations. It is a high-throughput detection method that is ready for application in clinical laboratories.

Population genetics of four PKLR intragenic polymorphisms in Portugal and São Tomé e Príncipe (Gulf of Guinea).

Four intragenic PKLR polymorphisms [1705A/C, 1738C/T. T10/19, and (ATT)n microsatellite] were studied in normal population samples of Central Portugal and São Tomé e Príncipe, a small archipelago located in the Gulf of Guinea, West Africa. For all loci, the observed genotype distributions do not deviate from Hardy-Weinberg equilibrium. The allele frequencies found in the Portuguese population are similar to those previously described in Caucasian populations. Mother-child pair analysis for the (ATT)n microsatellite does not show deviations to the Mendelian rules. In São Tomé e Príncipe the biallelic polymorphisms 1705A/C, 1738C/T, and T10/19 presented inverse allelic frequencies when compared with the Portuguese population. Two new alleles were found at the (ATT)n microsatellite. Significant statistical differences were found between both populations. The results showed that São Tomeans had higher haplotype diversity and lower linkage disequilibrium among the polymorphic sites. The PKLR intragenic polymorphisms, commonly used in haplotype analysis with the gene mutations in PK-deficient patients, can thus be successfully employed in anthropological genetics.

A new PKLR gene mutation in the R-type promoter region affects the gene transcription causing pyruvate kinase deficiency.

Mutations in the PKLR gene responsible for pyruvate kinase (PK)-deficient anaemia are mainly located in the coding regions: 11 are in the splicing sites and, recently, three mutations have been described in the promoter region. We now report a novel point mutation A-->G on nucleotide 72, upstream from the initiation codon of the PKLR gene, in four Portuguese PK-deficient patients. This new regulatory mutation occurs within the most proximal of the four GATA motifs (GATA-A element) in the R-type promoter region. In two patients who were homozygous for this mutation, a semiquantitative reverse transcription polymerase chain reaction (PCR) procedure was used to evaluate the amount of R-PK mRNA transcript in the reticulocytes. The mRNA level was about five times lower than in normal controls, demonstrating that the PKLR gene transcription is severely affected, most probably because the -72A-->G point mutation disables the binding of the erythroid transcription factor GATA-1 to the GATA-A element. Supporting these data, the two patients homozygous for the -72A-->G mutation had severe haemolytic anaemia and were transfusion dependent until splenectomy. Two other patients who were compound heterozygous for this mutation and the previously described missense mutation 1456C-->T had a mild condition.

PK-LR gene mutations in pyruvate kinase deficient Portuguese patients.

In nine unrelated Portuguese patients with pyruvate kinase (PK) deficient anaemia, whose symptoms ranged from a mild chronic haemolytic anaemia to a severe anaemia presenting at birth and requiring multiple transfusions, the PK-LR gene mutations were identified and correlated with their phenotypes. Five different mutations were identified, three of them for the first time: a missense mutation 1670G --> C on exon 12 and two 5' splice donor site (GT) mutations on intron 8 [IVS8(+2)T --> G] and intron 10 [IVS10(+1)G --> C]. Two previously described missense mutations, 1456C --> T and 993C --> A, were also found. The genotype/phenotype correlation showed that patients with two missense mutations or with a missense mutation and a splicing mutation had a mild haemolytic anaemia. The three patients with severe anaemia, who were transfusion dependent until splenectomy, were homozygous for the splicing site mutations IVS10(+1)G --> C or IVS8(+2)T --> G.

Human erythrocyte pyrimidine 5'-nucleotidase isoenzymes: effect of sulfhydryl reagents and electrophoretic discrimination.

The electrophoretic behavior of human red cell pyrimidine 5'-nucleotidase isozymes (UMPH1 and UMPH2) was studied on starch gels with and without treatment with thiol reagents. It was found that at least one reactive sulfhydryl group occurs in the UMPH1 isozyme but not in the UMPH2 isozyme. An electrophoretic system is described that allows the discrimination of UMPH1 and UMPH2 isozymes.

Meniscal tears--comparison of arthrography, CT, and MRI.

A total of 1750 knees were prospectively evaluated using a high-resolution noninvasive axial computed tomography (CT) scanning technique. A total of 203 knees underwent subsequent arthroscopic or arthrographic evaluation. In this group, the sensitivity of CT for the detection of a torn meniscus was 88.5%; the specificity was 95.5%; and the accuracy was 91.5%. An additional 270 knees were prospectively evaluated by both CT and magnetic resonance imaging (MRI) to determine the ability of both techniques to characterize knee menisci in patients believed to have meniscal tears. Of these knees, 94 were subsequently examined by arthroscopy. In this group, the overall accuracy of MRI for detecting a torn meniscus was 89.5% and for CT it was 92.2%. Properly performed, both CT and MRI are accurate and effective methods for noninvasively evaluating meniscal abnormalities in the acutely injured knee. The protocol for CT and MRI meniscus imaging as well as interpretation are presented in addition to their relative roles with respect to arthrography and arthroscopy.

Bucket-handle tears of the meniscus: appearance at CT.

Before undergoing arthroscopy, 222 patients with acute, unilateral knee injuries and strong clinical evidence of a meniscal tear underwent axial computed tomography (CT) of the knee. In 53 patients, a bucket-handle meniscal tear was found, and it was repaired at arthroscopy. In 92.5% (49 of 53) of these knees, CT accurately depicted the type, location, and extent of the tear. CT also demonstrated the displaced meniscal fragment with its relationship to the attached remnant.

Noninvasive evaluation of knee meniscal tears: preliminary comparison of MR imaging and CT.

One hundred twenty knees were examined prospectively with both axial computed tomography (CT) and magnetic resonance (MR) imaging to compare the value of these techniques in patients with clinical evidence of meniscal tears. Sixty-four of these knees were subsequently evaluated with diagnostic arthroscopy. In this group, CT was superior to MR imaging for meniscus evaluation in 29.7% of the knees, equal to MR in 54.7%, and inferior to MR in 15.6%. Although surface-coil MR imaging shows great promise and has numerous advantages over more conventional techniques, this preliminary experience suggests that, at least with certain imaging equipment and techniques, CT may be slightly more efficacious than 0.5-T MR imaging in meniscus evaluation. However, further comparative studies at higher field strengths are needed before the relative roles of CT and MR imaging can be established.

Diagnosis of meniscal tears using high-resolution computed tomography. Correlation with arthroscopy.

Four hundred and fifty patients (840 knees) underwent axial computed tomography of the knee in a prospective study to evaluate the ability of this technique to identify and characterize meniscal tears. One hundred and thirty-three of the knees subsequently had diagnostic arthroscopy. When compared with the findings at arthroscopy, the sensitivity of computed tomography was 96.5 per cent, the specificity was 81.3 per cent, and the accuracy was 91.0 per cent. The precise protocol for the method of computed tomographic scanning that was employed and the criteria that were used to interpret the images are presented. We found computed tomography to be a valuable non-invasive method for the detection and characterization of meniscal tears.

Fetal small bowel simulating an abdominal mass at sonography.

A focal, well-marginated, homogeneous hyperechoic mass was identified within the fetal abdomen of five early gestations varying in age from 16 to 20 weeks ECA (estimated conceptual age). Initially, the possibility of a congenital abdominal tumor mass was raised. Serial sonography subsequently demonstrated progressive dissolution of the masses, each of which assumed the characteristic appearance of normal small bowel by 30 weeks ECA. The recognition of this normal fetal small bowel maturation pattern is important lest it be confused with congenital retroperitoneal or gastrointestinal tract abnormalities.

Meniscus tears of the knee: prospective evaluation with CT.

A total of 209 patients underwent prospective axial computed tomography (CT) examinations of the knee to evaluate the ability of this technique to identify and characterize knee menisci in patients believed to have meniscus tears. Of the 359 knees examined, 105 subsequently underwent arthrography, arthroscopy, or arthrography and arthroscopic surgery. In this group, the sensitivity of CT was 88.5%, specificity was 95.5%, and accuracy was 91.5%. Although axial CT is a sensitive and effective method for the detection and characterization of tears involving the medial and lateral menisci, purely horizontal or nondisplaced peripheral tears may be difficult to demonstrate.

Splint therapy evaluation with direct sagittal computed tomography.

Splint therapy is often the initial nonsurgical treatment selected to manage patients who have anterior displacement of the temporomandibular joint (TMJ) meniscus. It is usually effective if the splint restores normal meniscocondylar relationships. An accurate method for assessing meniscocondylar relationships by means of direct sagittal computed tomography (CT) is described. The precise protocol for this noninvasive CT method and the criteria used to interpret the CT images are presented.