Genetic testing improves the diagnosis of adult type hypolactasia in the Mediterranean population of Sardinia.

OBJECTIVE: Recently, the C/T-13910 polymorphism on chromosome 2q21 in North-European populations has been found completely associated with lactase activity and its genetic typing proposed as first-stage screening test for adult hypolactasia. However, the C/T-13910 variant in some sub-Saharan African groups is not a predictor of lactase persistence. In this work, we wanted to verify if in the Mediterranean island of Sardinia, located in Southern Europe, the C/T-13910 polymorphism may be useful or not for the diagnosis of adult type hypolactasia. DESIGN: Validation study of a genetic testing for adult type hypolactasia in Sardinians. SETTING: Brotzu Hospital and Microcitemico Hospital, Cagliari, Italy. SUBJECTS: The sample consisted in 84 Sardinian individuals (63 women and 21 men; range 20-73 years) selected from a group of 832 patients. METHODS: Genetic testing was compared to an improved test obtained by a combination of different breath hydrogen tests and clinical assessment. RESULTS: We found that all 49 individuals with lactose malabsorption, demonstrated by a combination of different breath hydrogen tests and clinical assessment, carried the C/C-13910 genotype associated with lactase non-persistence, 23 individuals with lactose normal absorption carried the C/T-13910 genotype associated with lactase persistence and only one person with the above phenotype showed a discordant C/C-13910 genotype. The genetic testing showed very high sensitivity, specificity, positive and negative predictive values of 100, 95.8, 98 and 100%, respectively. CONCLUSIONS: Sardinians, unlike some ethnic groups in sub-Saharan Africa, show the same genetic association of hypolactasia with the C/T-13910 variant as other North-European populations. The genetic testing for the C/T-13910 variant may contribute to improving the diagnosis of adult type hypolactasia.

The HLA-DPB1--associated component of the IDDM1 and its relationship to the major loci HLA-DQB1, -DQA1, and -DRB1.

The major histocompatibility complex (MHC) HLA region on chromosome 6p21 contains the major locus of type 1 diabetes (IDDM1). Common allelic variants at the class II HLA-DRB1, -DQA1, and -DQB1 loci account for the major part of IDDM1. Previous studies suggested that other MHC loci are likely to contribute to IDDM1, but determination of their relative contributions and identities is difficult because of strong linkage disequilibrium between MHC loci. One prime candidate is the polymorphic HLA-DPB1 locus, which (with the DPA1 locus) encodes the third class II antigen-presenting molecule. However, the results obtained in previous studies appear to be contradictory. Therefore, we have analyzed 408 white European families (200 from Sardinia and 208 from the U.K.) using a combination of association tests designed to directly compare the effect of DPB1 variation on the relative predisposition of DR-DQ haplotypes, taking into account linkage disequilibrium between DPB1 and the DRB1, DQA1, and DQB1 loci. In these populations, the overall contribution of DPB1 to IDDM1 is small. The main component of the DPB1 contribution to IDDM1 in these populations appears to be the protection associated with DPB1*0402 on DR4-negative haplotypes. We suggest that the HLA-DP molecule itself contributes to IDDM1.

Cytochrome P450 1A2 is a hepatic autoantigen in autoimmune polyglandular syndrome type 1.

Autoantibodies directed against proteins of the adrenal cortex and the liver were studied in 88 subjects of Sardinian descent, namely six patients with autoimmune polyendocrine syndrome type 1 (APS1), 22 relatives of APS1 patients, 40 controls with other autoimmune diseases, and 20 healthy controls. Indirect immunofluorescence, using tissue sections of the adrenal cortex, revealed a cytoplasmatic staining pattern in 4 of 6 patients with APS1. Western blotting with adrenal mitochondria identified autoantigens of 54 kDa and 57 kDa, Western blotting with placental mitochondria revealed a 54-kDa autoantigen. The 54-kDa protein was recognized by 4 of 6 patients with APS1 both in placental and adrenal tissue, whereas the 57-kDa protein was detected only by one serum. Using recombinant preparations of cytochrome P450 proteins, the autoantigens were identified as P450 scc and P450 c17. One of six APS1 patients suffered from chronic hepatitis. In this patient, immunofluorescence revealed a centrolobular liver and a proximal renal tubule staining pattern. Western blots using microsomal preparations of human liver revealed a protein band of 52 kDa. The autoantigen was identified as cytochrome P450 1A2 by use of recombinant protein preparations. P450 1A2 represents the first hepatic autoantigen reported in APS1. P450 1A2 usually is not detected by sera of patients with isolated autoimmune liver disease and might be a hepatic marker autoantigen for patients with APS1.

Combinations of specific DRB1, DQA1, DQB1 haplotypes are associated with insulin-dependent diabetes mellitus in Sardinia.

The Sardinian population has an extremely high incidence of IDDM (30.2 of 100.000 in the age group of 0-14 years). This study reports the molecular characterization of HLA class II genes in 120 IDDM sporadic patients and 89 healthy subjects of Sardinian origin. Compared with other Caucasians, both Sardinian patients and controls had an unusual distribution of haplotypes and genotypes. In particular, there was a high gene frequency of the DRB1*0301, DQA1*0501, DQB1*0201 susceptibility haplotype both in patients (0.58) and controls (0.23) while a reduction of the DRB1*1501, DQA1*0102, DQB1*0602 protective haplotype (0.03) was observed in the healthy population. This distribution may partially explain the high incidence of IDDM reported in Sardinia. The analysis of the DQ beta 57 and DQ alpha 52 residues showed that the absence of Asp 57 and the presence of Arg 52 were associated with IDDM in a dose-response manner. On the other hand, we found that (a) a very similar distribution of these residues was found when comparing Sardinians with another healthy Caucasian population from the same latitude but with a lower rate of IDDM incidence; (b) several genotypes encoding the identical DQ alpha 52/DQ beta 57 phenotype carried very different relative risks; and (c) the DRB1*0403, DQA1*0301, DQB1*0304 haplotype (DQ beta 57 Asp-neg and DQ alpha 52 Arg-pos) was found in 40% of the DR4-positive controls but not in patients (p = 0.00034), while the DRB1*0405, DQA1*0301, and DQB1*0302 haplotype carrying the same residues at the same positions was found in 70% of the DR4-positive patients and in only one control (p = 0.00003). These findings suggest that IDDM susceptibility cannot be completely explained by the model in which only DQ alpha 52 and DQ beta 57 residues are taken into account.

The distribution of DR4 haplotypes in Sardinia suggests a primary association of type I diabetes with DRB1 and DQB1 loci.

The contribution of genetic variation at HLA class II loci to the susceptibility to and protection from IDDM was investigated by analyzing the distribution of HLA-DRB1*04 haplotypes in 630 Sardinian newborns and 155 Sardinian IDDM patients. The different RRs and ARs of the various DR4-DQB1*0302 haplotypes, significantly ranging from the strongly associated DRB1*0405, DQB1*0302 to the protective DRB1*0403, DQB1*0302 haplotypes, provides clearcut evidence that the DRB1 locus is crucial in conferring IDDM predisposition or protection. Also, the DQB1 locus influences IDDM predisposition or protection by restricting the disease-positive association to DRB1*0405 haplotypes carrying the susceptibility DQB1*0302 or DQB1*0201 alleles but not the protective DQB1*0301 allele. Haplotype analysis not only suggests that the DRB1 and DQB1 loci influence IDDM risk in the same way, but also that the HLA-linked protection is "dominant" compared with "susceptibility." These results, obtained from a population with one of the highest IDDM incidences in the world, define more clearly the contribution of the various HLA loci to IDDM protection or susceptibility and allow a more precise calculation of AR.

A gene dosage effect of the DQA1*0501/DQB1*0201 allelic combination influences the clinical heterogeneity of celiac disease.

This study reports the HLA-DR and DQ molecular characterization of 62 CD patients of Sardinian descent. Patients were divided in two groups (36 in group I and 26 in group II) according to the clinical features at the disease onset. Among the patients of group I, having the fully expressed form of CD and a mean age of 3 years at disease onset, a significant increase of DRB1*0301, DQA1*0501, DQB1*0201 homozygotes, encoding in cis two DQ (alpha 1*0501, beta 1*0201) susceptibility heterodimers, was observed when compared either with the patients of group II (pIII < 0.012) or with healthy individuals (pI < 10(-6)). On the other hand, in the patients of group II, presenting oligosymptomatic forms and a mean age of 5.7 years at the disease onset, the haplotype combinations encoding in cis or in trans only one DQ (alpha 1*0501, beta 1*0201) heterodimer were significantly increased in comparison either with the patients of group I (pIII < 0.026) or with controls (pII < 10(-6)). These findings suggest that a double dose of DQA1*0501, DQB1*0201 genes may predispose a person to an earlier onset and to more severe disease manifestations. Genotype analysis showed that only three patients (all in group I) failed to form in trans or in cis the DQ (alpha 1*0501, beta 1*0201) heterodimer and carried the DQA1*0101,DQB1*0501 haplotype, suggesting its possible role in CD susceptibility. In addition, a significant increment of DQB1*0501 gene (pc < 0.0065) was found comparing the frequency of DQB1 alleles in CD patients and healthy controls, after exclusion of DQB1*0201 chromosomes.(ABSTRACT TRUNCATED AT 250 WORDS)

HLA-DQB1*0305 and -DQB1*0304 alleles among Sardinians. Evolutionary and practical implications for oligotyping.

This study, performed in individuals of Sardinian descent, reports an epidemiologic and molecular analysis of the recently identified DQB1*0304 and DQB1*0305 alleles. These two alleles having a gene frequency of 0.017 and 0.005, respectively, are not uncommon in Sardinia and are distributed fairly uniformly on the island. The analysis of DQB1 second and third exons of the two alleles revealed that although they have always been found included within the same DRB1*0403-DQA1*03 haplotype, they had a different origin. The sequence pattern of DQB1*0305 confirmed that it originated from the DQB1*0302 "recipient" gene by the insertion of a DQB1*0402 nucleotide stretch, within its beta-sheet region, while that of DQB1*0304 suggested that it originated from the DQB1*0301 gene, either by a single point mutation at codon 57 (GCC instead of GAC) or, alternatively, by a segmental transfer of a DQB1*0302 motif, including codon 57, within its alpha-helic region. Independently from the mechanism of generation, the fact that DQB1*0304 originated from DQB1*0301 allele was intriguing considering that, in over 1500 HLA class II Sardinian haplotypes examined, neither the putative parental DRB1*0403-DQA1*03-DQB1*0301 haplotypes were found. Finally, since the assignment of DQB1*0305 may be inaccurate with the traditional panel of probes commonly used for DQB1 oligotyping, the use of an additional oligonucleotide probe is recommended.

Haematological characteristics of the beta 0 thalassaemia trait in Sardinian children.

We report red cell indices and haemoglobin (Hb)A2 levels in Sardinian children with heterozygous beta 0-thalassaemia and in normal controls aged 6 months to 12 years. Iron-deficient children and those with haematological findings indicative of alpha-thalassaemia were excluded. As in adult carriers, these subjects have significantly increased mean red cell counts and significantly reduced mean Hb levels, mean corpuscular volume (MCV), mean corpuscular haemoglobin (MCH), haematocrit, and mean corpuscular haemoglobin concentration. From 66 to 76% of the heterozygous beta 0 thalassaemia children examined were anaemic. MCH and MCV were within the normal range in 2.8% of these children. Serum ferritin levels showed no difference from those of normal controls.

Chronic liver disease in transfusion-dependent thalassaemia: hepatitis B virus marker studies.

The systematic screening of 253 children with transfusion-dependent homozygous beta-thalassaemia revealed a high incidence of hepatitis B virus markers. The highest frequencies of hepatitis B surface antigen (HBsAg) and antibody to hepatitis B core antigen (anti-HBc) were found in the group of patients with the smallest number of transfusions, while the highest frequency of antibody to hepatitis B surface antigen (anti-HBs) was detected in the patients who had had the largest number of transfusions. Follow-up of these patients showed (a) a high incidence of acute hepatitis B, which was mainly subclinical; (b) normal hepatitis B surface antigen clearance and normal antibody to hepatitis B surface development; and (c) a high frequency of increased transaminase values for over six months. In all the subjects with persistently high transaminase, histological examination revealed chronic persistent hepatitis or chronic active hepatitis. Apart from two cases of chronic active hepatitis with no B virus markers, and two cases of chronic persistent hepatitis with HBsAg and anti-HBc in the serum, all these subjects were anti-HBs positive but HGsAg and anti-HBc negative.

Acute lymphoblastic leukemia in a child with constitutional ring chromosome 21.

This report describes a case of acute lymphoblastic leukemia with non-B, non-T, common acute lymphocytic leukemia antigen-positive blasts in a 13-year-old child with constitutional ring chromosome #21. Because ring chromosome #21 is a rare chromosomal disorder, it is likely that the leukemia transformation is related to the chromosomal anomaly.

Schwartz-Jampel syndrome. Clinical, electrophysiological and histopathological study of a severe variant.

This report describes a child, offspring of a first cousin marriage, with a severe and progressive disorder of bone and cartilage growth associated with a myotonia-like syndrome. Clinical manifestations of this disease began at birth with marked generalized muscle hypertrophy, stiffness, myotonia and multiple skeletal deformities. Successively severe dwarfism and mental retardation were observed. Neurophysiological studies showed continuous high frequently low voltage activity at rest and myotonic discharges which did not wax and wane. These abnormalities persisted after complete curarization. Muscle biopsy showed mild dystrophic changes. Polymeric glycosaminoglycans and degradation product excretion was normal. These findings are similar to those described in Schwartz-Jampel syndrome, but since the clinical picture was fully expressed at birth and was unusually severe, it is suggested that the patient's condition may have represented a severe variant of this syndrome.

Autoantigen characterization in liver/kidney microsome positive hepatitis.

Liver/kidney microsome autoantibodies are detectable in different forms of chronic hepatitis, namely autoimmune, viral, and drug-induced hepatitis and in hepatitis associated with Type 1 autoimmune polyglandular syndrome. Based on the aetiology of chronic hepatitis, liver/kidney microsome autoantibodies are directed against different enzymes with very little overlap. Thus, the simple Indirect Immunofluorescence test, which is universally used as a screening test to detect autoantibodies, does not allow subtyping of liver/kidney microsome autoantibodies. This brief review stresses the need to use methods such as Western-Blotting and enzyme-linked immunosorbent assay together with Indirect Immunofluorescence to characterize the liver/kidney microsome autoantibodies. Identification of the liver/kidney microsome target antigens, when possible, makes differential diagnosis easier and, at times, may help the clinician to choose the best approach to treatment.

Fatal Yersinia enterocolitica meningitis in thalassemia major.

A case of meningitis by Yersinia enterocolitica in a patient with thalassemia major is described. The Yersinia has been identified both in the cerebrospinal fluid and in the feces. The clinic syndrome began with gastroenteritis and was suddenly complicated by septicemia and meningitis. In spite of a prompt and specific antibiotic therapy, the disease led the patient to death.

The distribution of HLA class II haplotypes reveals that the Sardinian population is genetically differentiated from the other Caucasian populations.

In this study we have established the frequencies of the DRB1-DQA1-DQB1 haplotypes in a large cohort of Sardinian new-borns and found that the most frequent haplotypes were detected at frequencies unique to the Sardinians. Other haplotypes, common in other Caucasian populations, are rare or absent across the island. Next, the DRB1-DQA1-DQB1 haplotype frequencies obtained in Sardinians and those reported in other human populations were used to compute genetic distances and construct phylogenetic trees. A clear-cut pattern appeared with a split between the three major human groups: Caucasians, Asians and Blacks. Among the Caucasians there were three major clusters: a group representing the North-Africans, a group including most of the European-derived populations and a group encompassing Bulgaria, Greece and Sardinia. When we increased the resolution of the tree using the genetic distances calculated from both DRB1-DQA1-DQB1 haplotypes and class I HLA A, B, C allelic frequencies, the Sardinians clearly emerged as the major outlier among the various European populations considered in this study. These results indicate that the genetic structure of the present Sardinian population is the result of a fixation of haplotypes, which are very rare elsewhere, and are most likely to have originated from a relatively large group of founders.

Serum ferritin levels in hemoglobin H disease.

This study shows that hemoglobin H disease patients aged between 0.5 and 44 years, usually (27 out of 30) have normal serum ferritin levels according to age. This reconfirms that in this disease there are usually normal iron stores. However, in a few patients (3 out of 30) increased levels were found. This may be due to inappropriate iron medication, transfusions or associated idiopathic hereditary hemocromatosis gene.

Interaction between the glucose-6-phosphate dehydrogenase deficiency and thalassaemia genes at phenotype level.

No significant differences were observed in the mean values of Hb A2 levels and red cell indices between G6PD- and G6PD+ beta thalassaemia carriers apart from the MCV, which was significantly higher in beta thalassaemia G6PD- subjects, but still in the thalassaemia carrier range. No difference was seen between G6PD+ and G6PD- alpha thalassaemia carriers. G6PD+ beta thalassaemia carriers show a significant increase in G6PD levels expressed as activity per g of Hb and to lesser extent as activity per number of red cells x 10(9); in G6PD+ alpha thalassaemia carriers this increase is statistically significant only when the enzyme levels are expressed as activity per g of Hb. G6PD- beta thalassaemia carriers had enzyme levels higher than non-thalassaemic G6PD- subjects only when the activity is expressed per g of Hb. G6PD activity was found to be increased in G6PD+ and G6PD- Hb H disease patients.