Autoantibodies against CYP-2C19: A Novel Serum Marker in Pediatric De Novo Autoimmune Hepatitis?

Diagnosis of de novo autoimmune hepatitis (AIH) after orthotopic liver transplantation (OLT) is challenging especially in the absence of hyper-γ-globulinemia. Circulating autoantibodies are not sensitive nor specific in de novo AIH but when positive increase the diagnostic probability. We report the discovery of novel liver microsomal (LM) autoantibodies against CYP-2C19 in a 9-year-old boy with "de novo" AIH developed 7 years after OLT. Graft dysfunction presented with hypertransaminasemia (up to 400 IU/L), while serum γ-globulins remained within the normal range for age. Liver histology and response to high dose prednisone (2 mg/kg/day) with the addition of azathioprine therapy further supported the diagnosis of de novo AIH. Autoantibodies investigation by indirect immunofluorescence (IF) on rodent tissues showed a novel staining pattern involving the pericentral liver zone and sparing the renal tissue. Human but not rat liver proteins immunoblotting allowed us to characterize the novel LM antibodies and to identify CYP-2C19 as human antigen. The finding offers insights into the controversial discussion about autoimmunity versus alloreactivity with regard to the pathogenesis of de novo AIH. Correct information on human versus rat tissue antigens tested by methods other than IF for antibodies detection may have significant implications for the correct diagnosis and management of patients followed up after OLT.

Distinctive HLA-II association with primary biliary cholangitis on the Island of Sardinia.

BACKGROUND: The HLA DRB1*08 allele associated with primary biliary cholangitis (PBC) among Caucasians is of low frequency in the Sardinian population. OBJECTIVE: The aim of our study was to type a cohort of PBC patients from the island of Sardinia for HLA class II antigens. METHODS: Twenty Sardinian patients affected by PBC, 14 with autoimmune hepatitis (AIH) and 89 healthy controls (HCs) were typed for HLA class II alleles by dot-blot analysis. RESULTS: The PBC-associated HLA DRB1*08 allele was detected in none of the studied individuals. The DRB1*0301-DQB1*0201 was the prevalent HLA haplotype, detected in 19 (47.5%) out of 40 PBC haplotypes (OR = 3.0; 95% CI 1.5-6.2) and in 11 (39.3%) out of 28 AIH haplotypes (OR = 2.2; 95% CI 0.94-5.0), but in only 41 (23%) out of 178 HC haplotypes. Moreover, PBC patients showed an increased frequency of homozygosity for the DQB1*0201 allele (35% compared with 6.7% of the HCs; OR = 7.5; 95% CI 2.2-25.7). The frequency of the DRB1*11 allele in the PBC group was about half of that seen in the Sardinian HCs (7.5% vs 15.7%) (p = ns). CONCLUSIONS: Our study confirmed the low frequency of the HLA DRB1*08 allele among Sardinians, either in the general population or PBC patients. The high prevalence of the HLA DRB1*0301-DQB1*0201 haplotype is a distinctive genetic feature of PBC among Sardinians. Our study strengthens the hypothesis that still unknown genetic, epigenetic, and environmental factors must be involved in the pathogenesis of different HLA-associated liver diseases, and it represents a pathfinder that warrants exploration in a future extensive study.

Wilson's disease in two consecutive generations: the detection of three mutated alleles in the ATP7B gene in two Sardinian families.

BACKGROUND: Wilson's disease diagnosis is still a challenge for clinicians. AIM: To underline the importance of genetic testing in carrier detection and diagnosis of atypical Wilson's disease cases. METHODS: Two families with Wilson's disease in two consecutive generations were analysed with clinical, biochemical and genetic testing. RESULTS: In one family with triplet siblings, two of whom monozygotic, molecular screening of ATP7B, the gene responsible for Wilson's disease phenotype, allowed detection of 3 disease alleles, the discrimination between carrier and disease state and the postmortem diagnosis of Wilson's disease in the siblings' father. In the second family, molecular analysis detected 3 disease alleles and confirmed the diagnosis of Wilson's disease in two asymptomatic monozygotic twins. CONCLUSION: These results demonstrate that mutational analysis is determinant for carrier identification and diagnosis of atypical Wilson's disease patients.

Feasibility of RNA studies on illegitimate transcription for molecular characterization of splicing mutations in the ATP7B gene: a case report.

Approximately 520 Wilson disease-causing mutations in the ATP7B gene have been described to date. In this study we report DNA and RNA analyses carried out for molecular characterization of a consensus sequence splicing mutation found in homozygosity in a Swiss Wilson disease patient. RNA analysis of 1946 +6 T→C in both the peripheral lymphoblasts and liver resulted in the production in the propositus of only an alternative transcript lacking exons 6, 7, and 8 resulting most likely in alterations of cell biochemistry and disease. The patient presents an early form of severe hepatic disease characterized by hepatosplenomegaly, reduced hepatic function, anemia and thrombocytopenia indicating that 1946 +6 T→C is a severe mutation. Since identical results were obtained from both peripheral lymphoblasts and liver they also suggest that RNA studies of illegitimate transcripts can be safely used for molecular characterization of ATP7B splicing mutations, thus improving genetic counseling and diagnosis of Wilson disease. Moreover these studies, contribute to reveal the exact molecular mechanisms producing Wilson disease.

High frequency of low-risk human leukocyte antigen class II genotypes in latent celiac disease.

Human leukocyte antigen (HLA) class II genotypes in latent celiac disease, a clinical variant of celiac disease (CD) have been scarcely studied. The aim of this work was to investigate whether latent CD and CD share similar frequencies of HLA class II genotypes. HLA class II genotypes of CD patients compared with controls were subdivided in the following at-risk groups: DQB1*02/DQB1*02 (43.0%, odds ratio [OR] 8.02, p < 0.0001), DQB1*0302/DQB1*02 (12.2%, OR 2.77, p = 0.0002), DQB1*02/DQB1*X (39.2%, OR 1.23, p = 0.1903), DQB1*0302/DQB1*X (3.4%, OR 0.35, p = 0.0064) and DQB1*X/DQB1*X (0.8%, OR 0.01, p = 0.0001) where X is neither DQB1*0302 nor DQB1*02. Next, HLA class II genotypes of 21 latent CD patients were compared with the above at-risk groups. Only one latent CD patient (4.8%) was found in the high risk DQB1*02/DQB1*02 group, three (14.3%) were DQB1*0302/DQB1*02, one (4.8%) was DQB1*0302/DQB1*X and the remaining 16 (76.2%) showed the DQB1*02/DQB1*X genotype. Noteworthy, the only 1 patient with the DQB1*02/DQB1*02 high risk genotype did not carry the DR3-DQB1*02/DR3-DQB1*02 or the DR3-DQB1*02/DR7-DQB1*02 but the uncommon DR3-DQB1*02/DR4-DQB1*02 genotype. These data suggest that latent CD is prevalently associated with low-risk HLA class II genotypes.

Development of TaqMan allelic specific discrimination assay for detection of the most common Sardinian Wilson's disease mutations. Implications for genetic screening.

Wilson's disease (WD) is an autosomal recessive disorder caused by a defective function of the copper transporting ATP7B protein. Analysis of ATP7B gene in the Sardinian population revealed the presence of six common mutations that together account for 85% of WD chromosomes. We have developed an automated approach for the detection of these 6 common Sardinian mutations based on TaqMan technology. Ten DNA samples of WD patients carrying different combinations of the six most common Sardinian mutations and normal controls previously analysed were used in triplicate to set up the allelic discrimination assays. The system was validated in 96 samples obtained from WD patients carrying different combinations of the most common mutations under investigation. The results showed that allelic discrimination is a valid method that could be used for efficient diagnosis of single cases but also for a mass screening.

RNA analysis of consensus sequence splicing mutations: implications for the diagnosis of Wilson disease.

Wilson disease (WD) is an autosomal recessive disorder caused by a defective function of the copper-transporting ATP7B protein. This results in progressive copper overload and consequent liver, brain, and kidney damage. Approximately 300 WD-causing mutations have been described to date. Missense mutations are largely prevalent, while splice-site mutations are rarer. Of these, only a minority are detected in splicing consensus sequences. Further, few splicing mutations have been studied at the RNA level. In this study we report the RNA molecular characterization of three consensus splice-site mutations identified by DNA analysis in WD patients. One of them, c.51 + 4 A --> T, resides in the consensus sequence of the donor splice site of intron 1; the second, c. 2121 + 3 A --> G, occurred in position + 3 of intron 7; and the c.2447 + 5 G --> A is localized in the consensus sequence of the donor splice site of intron 9. Analysis revealed predominantly abnormal splicing in the samples carrying mutations compared to the normal controls. These results strongly suggest that consensus sequence splice-site mutations result in disease by interfering with the production of the normal WD protein. Our data contribute to understanding the mutational spectrum that affect splicing and improve our capability in WD diagnosis.

High incidence and allelic homogeneity of Wilson disease in 2 isolated populations: a prerequisite for efficient disease prevention programs.

OBJECTIVES: Herein we report the results of mutation-based screening for Wilson disease (WD) in 2 isolated populations of Sardinia and the Greek island of Kalymnos. PATIENTS AND METHODS: Mutation analysis was performed in 110 and 9 WD families originating respectively from Sardinia and Kalymons using single-strand conformation polymorphism and sequencing methods. In Sardinia, a limited screening was performed for -441/-427del in 5290 newborns, whereas in Kalymnos 397 newborns underwent mutation screening for H1069Q and R969Q using appropriate methods. RESULTS: In Sardinia, mutation analysis showed the presence of 6 mutations accounting for 85% of chromosomes, 1 of which (-441/-427del) is present in 61.7% of alleles. The screening for -441/-427del in 5290 newborns revealed the presence of 122 heterozygotes, which is equal to an allelic frequency of 1.15%. Assuming the same distribution of WD mutations in the general Sardinian population, we also inferred an allelic frequency of 0.77% for mutations other than -441/-427del, which accounts for an overall frequency of any WD mutation of 1.92%. Assuming Hardy-Weinberg equilibrium, these data could be translated into a WD incidence of 1 in 2707 live births. In Kalymnos, mutation analysis in 9 WD families revealed the presence of only 2 mutations. The screening of 397 newborns revealed the presence of 18 heterozygotes for H1069Q, 9 for R969Q, and 1 compound heterozygote for these mutations, which is equal to an allele frequency of 3.7%. Assuming Hardy-Weinberg equilibrium, the expected carrier rate is 7%. CONCLUSIONS: These data indicate the need for health education for WD prevention in these isolated populations.

Decline of lactase activity and c/t-13910 variant in Sardinian childhood.

OBJECTIVES: Our study aims to determine the age of onset of adult-type hypolactasia in Sardinians, and to establish the age at which genotyping of the C/T-13910 variant can be used reliably in the diagnosis of lactose malabsorption. PATIENTS AND METHODS: A lactose breath hydrogen test was given to 383 randomly selected patients, from 3 to 19 years old. RESULTS: The C/C-13910 genotype was found in 90% of patients; the frequency of the positive lactose breath hydrogen test increased with age and reached a prevalence of 85% at 9 years. CONCLUSIONS: In Sardinians, adult-type hypolactasia becomes phenotypically evident in all individuals older than 9 years, suggesting that this should be considered the minimum age at which the genetic test for lactase nonpersistence should be applied.

Twenty-four novel mutations in Wilson disease patients of predominantly Italian origin.

Herein we report the results of mutation analysis of the ATP7B gene in a group of 134 Wilson disease (WD) families (268 chromosomes) prevalently of Italian origin. Using the SSCP and sequencing methods we identified 71 disease-causing mutations. Twenty-four were novel, while 19 more mutations already described, were identified in new populations in this study. A known mutation G591D showed a regional distribution, since it was only detected in 38.5% of the analyzed chromosomes in WD patients originating from Apulia, a region of South Italy. Detection of new mutations in the ATP7B gene increases our capability of molecular analysis that is essential for early diagnosis and treatment of WD.

Analysis of the T1288R mutation of the Wilson disease ATP7B gene in four generations of a family: possible genotype-phenotype correlation with hepatic onset.

Wilson disease, an autosomal recessive disorder due to mutations of the ATP7B gene, is characterized by copper accumulation and toxicity in the liver and subsequently in other organs, mainly the brain and cornea. A new missense mutation (T1288R) of the ATP7B gene has recently been discovered in a Wilson disease patient in our laboratory. The aim of the present study was to analyze clinical and genetic features of more generations of the family of the patient in which the new mutation T1288R was discovered. A total of 19 subjects were studied; in particular, four generations of the patient's family were analyzed. The ATP7B gene was analyzed by single-strand conformational polymorphism followed by direct sequencing. Two brothers presented a clinical diagnosis of Wilson disease with an hepatic phenotype and a genotype characterized by the homozygotic mutation T1288R. The heterozygotic mutation T1288R was found in seven subjects belonging to all four generations. The present study represents the first screening for a Wilson disease mutation through four generations of a nonconsanguineous family. All the patients with the homozygotic T1288R mutation in the present pedigree presented an hepatic phenotype without a neurological presentation. Consequently, a genotype-phenotype correlation could be hypothesized, although further studies are necessary to clarify this topic.

HLA-DQB1*0201 homozygosis predisposes to severe intestinal damage in celiac disease.

OBJECTIVE: Celiac disease (CD) is a T-lymphocyte-mediated small intestinal enteropathy triggered and maintained by dietary gluten, with a strong genetic component mapping to the HLA genes encoding for the class II DQ(alpha1*0501, beta1*02) molecule. Damage of the small intestine may cause a variety of clinical signs ranging from isolated long-standing iron-deficiency anemia refractory to iron supplementation to forms of severe malnutrition that may become life threatening. However, patients carrying the typical intestinal lesions of CD and presenting no symptoms at all (silent CD) are also a common clinical observation. Since it is commonly assumed that clinical signs and symptoms tend to correlate with the severity of the intestinal damage, the purpose of this study was to investigate whether particular HLA class II genotypes might also influence the extent of intestinal damage and consequently the clinical presentation of the disease. MATERIAL AND METHODS: We retrospectively compared histological grading of celiac disease intestinal biopsies with HLA haplotype, age at onset of disease and clinical signs and symptoms. RESULTS: Our findings showed that homozygosis for the DQB1*0201 allele is associated with a higher severity of the histological score (p<0.008). Of note for the clinician, this work also suggests that the same type 3c of intestinal damage causes a different clinical syndrome, depending on the patient's age. CONCLUSIONS: The genetic predisposition at the HLA-DQB1 locus influences the severity of the mucosal damage in a dose-dependent manner, but not the clinical presentation, of celiac disease.

The canine copper toxicosis gene MURR1 is not implicated in the pathogenesis of Wilson disease.

BACKGROUND: It has recently been demonstrated that the Wilson disease (WD) protein directly interacts with the human homolog of the MURR1 protein in vitro and in vivo, and that this interaction is specific for the copper transporter. The aim of the present study was to clarify the role of MURR1 in the pathogenesis of WD as well as in other WD-like disorders of hepatic copper metabolism of unknown origin. METHODS: Using the single-strand conformation polymorphism (SSCP) method followed by sequencing, we analyzed the 5' untranslated region (UTR) and three exons of the MURR1 gene in three groups of patients: 19 WD: patients in whom no mutations were detected in the ATP7B gene, 53 WD: patients in whom only one mutation in the ATP7B gene was found, and 34 patients in whom clinical and laboratory data suggested a WD-like disorder of hepatic copper metabolism of unknown origin. RESULTS: We detected in these patients six rare nucleotide substitutions, namely one splice-site consensus sequence and one missense and four silent nucleotide substitutions. All substitutions except one were found in the heterozygous state. No difference in the frequencies of the various substitutions was observed between patients and controls. CONCLUSIONS: These data suggest that the MURR1 gene and its protein product are unlikely to play a primary role in the pathogenesis of Wilson disease. More extensive studies with larger numbers of clinically homogeneous patients should be carried out to establish whether nucleotide alterations in the MURR1 gene may have a role in causing WD or WD-like disorders or act as modifying factors in the phenotype variability in WD.

Antitissue transglutaminase antibodies outside celiac disease.

BACKGROUND: Tissue transglutaminase enzyme-linked immunosorbent assay (tTG-ELISA) has recently been proposed as a simple and fast screening test for celiac disease (CD). The rate of false-positive and false-negative tests with tTG-ELISA, however, has not been definitively established. Therefore, the aim of our study was to investigate anti-tTG antibodies (TGA) not only in untreated patients with CD and in healthy controls, but also in a large group of patients with other autoimmune diseases. METHODS: The presence of TGA was investigated in sera from 111 patients with untreated CD, 96 patients with other autoimmune conditions (28 with autoimmune liver disease, 46 with insulin-dependent diabetes mellitus, 10 with inflammatory bowel syndrome, 12 with type 1 polyglandular syndrome) and from 100 healthy controls using guinea pig tTG-ELISA (gp-TG/ELISA) and highly purified recombinant human tTG-ELISA (h-TG/ELISA). Western blotting with guinea pig tTG was also performed. RESULTS: Ninety-four patients with CD who tested positive for antiendomysial antibodies (AEA) and one who tested negative for AEA showed antibodies against the gp-TG. Among the controls, 50% of patients with autoimmune liver disease and 6.5% of patients with insulin-dependent diabetes mellitus tested positive with gp-TG/ELISA. Western blotting experiments revealed that the high rate of positive tests observed using ELISA among the control group sera is attributable to impurities in the gp-TG preparation. However, h-TG/ELISA tests were positive for the sera from all patients who tested positive for AEA and from one control who tested negative for AEA, whereas h-TG/ELISA tests were negative for all CD patients who tested negative for AEA and for other controls who tested negative for AEA. CONCLUSIONS: The frequency of false-negative and false-positive tests represents the major limit to the use of gp-tTG/ELISA. However, because h-TG/ELISA is both simple and fast, it could be used in large screening programs for CD.