An atypical Dent's disease phenotype caused by co-inheritance of mutations at CLCN5 and OCRL genes.

Dent's disease is an X-linked renal tubulopathy caused by mutations mainly affecting the CLCN5 gene. Defects in the OCRL gene, which is usually mutated in patients with Lowe syndrome, have been shown to lead to a Dent-like phenotype called Dent disease 2. However, about 20% of patients with Dent's disease carry no CLCN5/OCRL mutations. The disease's genetic heterogeneity is accompanied by interfamilial and intrafamilial phenotypic heterogeneity. We report on a case of Dent's disease with a very unusual phenotype (dysmorphic features, ocular abnormalities, growth delay, rickets, mild mental retardation) in which a digenic inheritance was discovered. Two different, novel disease-causing mutations were detected, both inherited from the patient's healthy mother, that is a truncating mutation in the CLCN5 gene (A249fs*20) and a donor splice-site alteration in the OCRL gene (c.388+3A>G). The mRNA analysis of the patient's leukocytes revealed an aberrantly spliced OCRL mRNA caused by in-frame exon 6 skipping, leading to a shorter protein, but keeping intact the central inositol 5-phosphatase domain and the C-terminal side of the ASH-RhoGAP domain. Only wild-type mRNA was observed in the mother's leukocytes due to a completely skewed X inactivation. Our results are the first to reveal the effect of an epistatic second modifier in Dent's disease too, which can modulate its expressivity. We surmise that the severe Dent disease 2 phenotype of our patient might be due to an addictive interaction of the mutations at two different genes.

A new deletion in 5'-end of dystrophin gene removing M and P promoters and dystrophin muscle enhancers.

We describe a 3-year-old boy who, at age of 8 months, during investigations for upper respiratory tract infection was found to have an incidental grossly elevated CK of 20,000 UI/l. Investigations showed only mild calf hypertrophy and absent Gower's sign, normal cognitive function. Electromyography (EMG) showed myopathic features. Electrocardiography and echocardiography were normal. His muscle biopsy revealed myopathic features indicating Duchenne-type dystrophy. Immunohistochemistry for dystrophin N-terminal, C-terminal and mid-rod antibodies analysis showed the complete absence of dystrophin in the muscle fibers. Genetic studies showed a 141.1 Kb deletion removing muscle promoter, muscle exon 1, Purkinje promoter, Purkinje exon 1, dystrophin muscle enhancers similar to one previously reported in a DMD patient who exhibited some residual expression of dystrophin. The difference in dystrophin expression between these two patients might be due to the extension of deletions. The precise delimitation of the macrodeletion here described provides a better understanding of functional organization of the 5' end of the DMD gene.

Triplet-primed PCR is more sensitive than southern blotting-long PCR for the diagnosis of myotonic dystrophy type1.

Genetic testing of myotonic dystrophy type 1 (DM1) is very important because it enables the diagnosis and indicates the severity of the disease. Mutation analysis is based on the detection of the number of CTG triplets in the 3' untranslated region of the myotonic dystrophy protein kinase (DMPK) gene. Sometimes it could be complicated by the presence of different patterns of repeat interruptions in the 5' and 3' ends of the expanded alleles recently described in about 3% to 5% of patients. To make molecular diagnosis easier and faster, the use of triplet-primed PCR (TP-PCR) for the detection of expansions in DM1 and other dynamic mutation diseases was proposed. Here we present the results of a retrospective study performed by TP-PCR on 100 subjects previously analyzed by Southern blotting-long PCR.

Responsiveness to oral iron and ascorbic acid in a patient with IRIDA.

Mutations in TMPRSS6 gene cause iron-refractory iron deficiency anemia, a rare autosomal recessive disorder characterized by hypochromic microcytic anemia not responsive to oral iron therapy and partially responsive to parenteral iron administration. Here we report a female infant homozygous for a loss of function mutation in TMPRSS6 gene, who responded to oral iron therapy when supplemented with ascorbic acid.

Iron-deficiency anemia secondary to mutations in genes controlling hepcidin.

The discovery of the peptide hormone hepcidin in 2001 has shed light on the control of iron metabolism. Studies in animal models over the past few years have demonstrated its key role in regulating iron homeostasis. It was found that hepcidin deficiency leads to iron overload, and that its overexpression leads to severe iron-deficiency anemia. Since then, other genes regulating hepcidin expression have been discovered, and defects in them mostly resulted in iron overload. In 2008, a new gene, TMPRSS6, was identified that encodes a negative regulator of hepcidin expression. This discovery has been of great relevance because TMPRSS6 is the first gene regulating hepcidin, mutations in which cause chronic iron-deficiency anemia. Recently, genome-wide association studies identified common TMPRSS6 variants associated with hematological parameters, suggesting that TMPRSS6 is crucial in the control of iron homeostasis and normal erythropoiesis.

Locus heterogeneity of Dent's disease: OCRL1 and TMEM27 genes in patients with no CLCN5 mutations.

Dent's disease is an X-linked renal tubulopathy caused by mutations mainly affecting the CLCN5 gene. Defects in the OCRL1 gene, which is usually mutated in patients with Lowe syndrome, have recently been shown to lead to a Dent-like phenotype, called Dent's disease 2. About 25% of Dent's disease patients do not carry CLCN5/OCRL1 mutations. The CLCN4 and SLC9A6 genes have been investigated, but no mutations have been identified. The recent discovery of a novel mediator of renal amino acid transport, collectrin (the TMEM27 gene), may provide new insight on the pathogenesis of Dent's disease. We studied 31 patients showing a phenotype resembling Dent's disease but lacking any CLCN5 mutations by direct sequencing of the OCRL1 and TMEM27 genes. Five novel mutations, L88X, P161HfsX167, F270S, D506N and E720D, in the OCRL1 gene, which have not previously been reported in patients with Dent's or Lowe disease, were identified among 11 patients with the classical Dent's disease phenotype. No TMEM27 gene mutations were discovered among 26 patients, 20 of whom had an incomplete Dent's disease phenotype. Our findings confirm that OCRL1 is involved in the functional defects characteristic of Dent's disease and suggest that patients carrying missense mutations in exons where many Lowe mutations are mapped may represent a phenotypic variant of Lowe syndrome.

C329X in KRIT1 is a founder mutation among CCM patients in Sardinia.

Cerebral cavernous malformations (CCMs) are CNS vascular anomalies associated with seizures, headaches and hemorrhagic strokes and represent 10-20% of cerebral lesions. CCM is present in 0.1-0.5 of the population. This disorder most often occurs sporadically but may also be familial. Familial cases are inherited as a dominant trait with incomplete penetrance and are estimated to account for KRIT1 10-40% of the patients. The identification of the genes involved in such disorders allows to characterize carriers of the mutations without clear symptoms. The first gene involved in CCM1 is KRIT1. In addition to two other genes have been described: MGC4607 (CCM2) and PDCD10 (CCM3). We selected 13 patients belonging to seven Sardinian families on the basis of clinical symptoms and Magnetic Resonance results. In MGC4607 gene an undescribed exon five deletion likely producing a truncated protein was identified in one family. In two patients with clear phenotype and in three asymptomatic relatives a 4 bp deletion in exon 9 of KRIT1 gene, leading to a premature stop codon, was detected. A unique nonsense mutation (C329X) has been found in seven patients and two asymptomatic subjects belonging to four unrelated families. Haplotype analysis revealed a common origin of this mutation. These data suggest a "founder effect" in Sardinia for the C329X mutation, similar to other mutations described in different populations.

Turner syndrome mosaicism: an unusual case with a de novo large dicentric marker chromosome: mos 45,X/46,X, ter rea(X;X)(p22.3;p22.3).

X/X translocations are quite rare in humans. The effect of this anomaly on the phenotype is variable and depends on the amount of deleted material and whether the chromosomes are joined by their long or short arms. We report an unusual case of Turner syndrome mosaicism in a 16-year-old girl, who was referred to our Institute for primary amenorrhoea associated with short stature. Endocrine evaluation revealed hypergonadotropic hypogonadism, which required a study of the karyotype. Cytogenetic analysis, performed on peripheral blood leucocytes, showed a mos 45,X/46,X,ter rea (X;X)(p22.3;p22.3) de novo karyotype. The prevalent cell line was 45,X (90% cells). A second cell line (10% cells) showed a very large marker chromosome, similar to a large metacentric chromosome. FISH (fluorescent in situ hybridisation) and molecular analysis revealed that the marker chromosome was dicentric and totally derived from the paternal X chromosome.

A mutation in the TMPRSS6 gene, encoding a transmembrane serine protease that suppresses hepcidin production, in familial iron deficiency anemia refractory to oral iron.

BACKGROUND: Hepcidin plays a key role in body iron metabolism by preventing the release of iron from macrophages and intestinal cells. Defective hepcidin synthesis causes iron loading, while overproduction results in defective reticuloendothelial iron release and iron absorption. DESIGN AND METHODS: We studied a Sardinian family in which microcytic anemia due to defective iron absorption and utilization is inherited as a recessive character. Five members showed iron deficiency anemia that was not responsive to oral iron and only partially responsive to parenteral iron administration. To investigate the involvement of known genes implicated in iron metabolism we carried out linkage analysis with microsatellite markers mapping close to these genes. Afterwards, a genome-wide search was performed. RESULTS: No linkage was found between the phenotype of the patients and several known human genes involved in iron metabolism (DMT1, TF, TFRC, ZIRTL, HAMP, HJV). Genome-wide scanning by microsatellites and single nucleotide polymorphisms showed a multipoint LOD score of 5.6 on chromosome 22q12.3-13.1, where the matriptase-2 (also known as transmembrane protease, serine 6 or TMPRSS6) gene is located. Its murine counterpart (Tmprss6) has recently been found to be an essential component of a pathway that detects iron deficiency and suppresses hepcidin production. Sequencing analysis of TMPRSS6 revealed a homozygous causal mutation, predicting a splicing error and a truncated TMPRSS6 protein in affected members. Homozygous subjects had inappropriately elevated levels of serum and urinary hepcidin. CONCLUSIONS: The findings of this study suggest that the observed TMPRSS6 mutation leads to overproduction of hepcidin and, in turn, to defective iron absorption and utilization. More generally, they confirm in humans the inhibitory effect of matriptase-2 on hepcidin synthesis already demonstrated in mice.

The Italian External Quality Assessment scheme for fragile x syndrome: the results of a 5-year survey.

The Italian External Quality Assessment scheme for fragile X syndrome started in 2001 as an activity funded by the National Health System and coordinated by the National Institute of Public Health. The aim of this work is to present the data of 5 years (2001--2004 and 2006) of survey. The External Quality Assessment scheme was designed to cover the following points: (a) genotyping and (b) interpretation and reporting of results. Overall, the scheme covered about 65% of all Italian public laboratories. The average reporting of results was 91.6%, with an overall success rate of 76%. The rate of diagnostic errors observed was on average 5%. Inaccuracy in sizing of CGG repeats of normal and premutated alleles was reported. During the survey the proportion of laboratories using a Southern blotting, polymerase chain reaction, and ABI sizing kit in combination rose from 36.8% to 70.6%. The reports from laboratories showed incompleteness and considerable variations in expected outcomes. For this reason, in 2004 a model for written reports was introduced. In conclusion, these data underscore the need to participate in External Quality Assessment schemes as an educational resource to ensure quality in molecular genetic testing.

All known patient mutations in the ASH-RhoGAP domains of OCRL affect targeting and APPL1 binding.

Mutations in the inositol 5-phosphatase OCRL are responsible for Lowe syndrome, an X-linked disorder characterized by bilateral cataracts, mental retardation, neonatal hypotonia, and renal Fanconi syndrome, and for Dent disease, another X-linked condition characterized by kidney reabsorption defects. We have previously described an interaction of OCRL with the endocytic adaptor APPL1 that links OCRL to protein networks involved in the disease phenotype. Here, we provide new evidence showing that among the interactions which target OCRL to membranes of the endocytic pathway, binding to APPL1 is the only one abolished by all known disease-causing missense mutations in the ASH-RhoGAP domains of the protein. Furthermore, we demonstrate that APPL1 and rab5 independently contribute to recruit OCRL to enlarged endosomes induced by the expression of constitutively active Rab5. Thus, binding to APPL1 helps localize OCRL at specific cellular sites, and disruption of this interaction may play a role in disease.

A novel interstitial deletion in Xq25, identified by array-CGH in a patient with Lowe syndrome.

The oculocerebrorenal syndrome of Lowe (OCRL) (MIM:309000) is an X-linked multisystemic disorder affecting the eyes, nervous system and kidneys due to mutations in OCRL1 gene. The gene contains 24 exons, and encodes a 105kDa phosphatydylinositol 4,5-biphosphate [PtdIns(4,5)P(2)] 5-phosphatase localized primarily in the trans-Golgi network and the lysosomes. The large majority of the OCRL1 mutations producing Lowe syndrome are either missense mutations localized mainly in the catalytic domain or non-sense/frameshift mutations resulting in truncated proteins. Rarely, in about 6% of the cases, the disease results from large gene deletions occurring in the 5' part of the gene. Here we report a new case of a patient with Lowe syndrome due to a deletion of about 4Mb, encompassing the OCRL1 gene, detected by PCR and CGH array. The mother was carrier of the same deletion.

A locus for familial skewed X chromosome inactivation maps to chromosome Xq25 in a family with a female manifesting Lowe syndrome.

In mammals, X-linked gene products can be dosage compensated between males and females by inactivation of one of the two X chromosomes in the developing female embryos. X inactivation choice is usually random in embryo mammals, but several mechanisms can influence the choice determining skewed X inactivation. As a consequence, females heterozygous for X-linked recessive disease can manifest the full phenotype. Herein, we report a family with extremely skewed X inactivation that produced the full phenotype of Lowe syndrome, a recessive X-linked disease, in a female. The X chromosome inactivation studies detected an extremely skewed inactivation pattern with a ratio of 100:0 in the propositus as well as in five out of seven unaffected female relatives in four generations. The OCRL1 "de novo" mutation resides in the active paternally inherited X chromosome. X chromosome haplotype analysis suggests the presence of a locus for the familial skewed X inactivation in chromosome Xq25 most likely controlling X chromosome choice in X inactivation or cell proliferation. The description of this case adds Lowe syndrome to the list of X-linked disorders which may manifest the full phenotype in females because of the skewed X inactivation.

OCRL mutation analysis in Italian patients with Lowe syndrome.

The oculocerebrorenal syndrome of Lowe (OCRL, also called OCRL1) is a rare X-linked disorder characterized by major abnormalities of eyes, nervous system, and kidneys. The gene responsible for OCRL was identified by positional cloning and encodes an inositol polyphosphate-5-phosphatase. We performed the molecular analysis in 9 Italian patients and 26 relatives and we detected the mutations in all the examined patients. Eight mutations out of nine had never been described and consisted of truncating mutations (frameshift, nonsense, splice site and genomic deletion), and missense mutations. The mutations were distributed in the second half of the gene as previously described in other populations. In three cases the mutations were absent in the mothers confirming the occurrence of novel mutations in this disorder. Our results on the Italian population are similar to the data previously obtained in other populations.

H63D mutation in the HFE gene increases iron overload in beta-thalassemia carriers.

BACKGROUND AND OBJECTIVES: Hereditary hemochromatosis (HH) is an autosomal recessive disorder of iron metabolism. The HFE gene implicated in this disorder has been identified on chromosome 6 (6p21.3). The most prevalent mutation in HH patients changes the 282 cysteine residue to tyrosine (C282Y). The role of a second mutation which changes the 63 histidine to aspartic acid (H63D) in iron overload has been controversial. The aim of this study was to evaluate the effect of the H63D mutation on the ferritin levels of beta-thalassemia carriers. DESIGN AND METHODS: beta-thalassemia carriers have a tendency to increase iron absorption because of mild anemia and slightly increased erythropoiesis. Differences in ferritin levels between homozygotes for H63D and wild type may indicate a modulator effect of the HFE mutation on iron absorption. We studied 152 healthy males, heterozygous for beta-thalassemia. Serum ferritin was measured by chemiluminescence. H63D genotypes were determined by digestion of polymerase chain reaction (PCR) products with MboI restriction enzyme. RESULTS: Forty-five subjects were H63D heterozygotes and four subjects were H63D homozygotes. Ferritin levels were (mean +/- SD): 250 +/- 138 microg/L in homozygotes for the wild type H/H; 295 +/- 186 microg/L in H/D heterozygotes; and 389 +/- 75 microg/L in homozygotes for the mutation D/D. The difference in ferritin values between H/H and D/D is statistically significant (p=0.022). INTERPRETATION AND CONCLUSIONS: beta-thalassemia carriers who are homozygotes for the H63D mutation have higher ferritin levels than beta-thalassemia carriers with the H/H genotype, suggesting that the H63D mutation may have a modulating effect on iron absorption.

Frequency of hemochromatosis C282Y and H63D mutations in Sardinia.

Hereditary hemochromatosis (HH) is one of the most common autosomal recessive disorders of iron metabolism among Caucasians, and it is associated with C282Y mutation of the HFE gene in populations of Celtic origins. A second mutation, H63D, shows a very high widespread frequency, although its role in iron metabolism is still inconclusive. There are no data on the frequencies of these two mutations in Sardinia, an island in the Mediterranean sea that has not been invaded by Celtic peoples. We examined 836 chromosomes from Sardinian subjects and tested for the mutation by restriction enzyme digestion of PCR products. Among the 836 analyzed chromosomes, we found a C282Y allele frequency of 0.0036 and an H63D allele frequency of 0.173. These data could explain the observed rarity of HH in Sardinia. The high allele frequency of H63D and the rarity of HH in Sardinia is suggestive that this mutation is not a major contributor to this disease.