Identification of MEFV-independent modifying genetic factors for familial Mediterranean fever.

Familial Mediterranean fever (FMF) is a recessively inherited disorder predisposing to renal amyloidosis and associated with mutations in MEFV, a gene encoding a protein of unknown function. Differences in clinical expression have been attributed to MEFV-allelic heterogeneity, with the M694V/M694V genotype associated with a high prevalence of renal amyloidosis. However, the variable risk for patients with identical MEFV mutations to develop this severe complication, prevented by lifelong administration of colchicine, strongly suggests a role for other genetic and/or environmental factors. To overcome the well-known difficulties in the identification of modifying genetic factors, we investigated a relatively homogeneous population sample consisting of 137 Armenian patients with FMF from 127 independent families living in Armenia. We selected the SAA1, SAA2, and APOE genes-encoding serum amyloid proteins and apolipoprotein E, respectively-as well as the patients' sex, as candidate modifiers for renal amyloidosis. A stepwise logistic-regression analysis showed that the SAA1alpha/alpha genotype was associated with a sevenfold increased risk for renal amyloidosis, compared with other SAA1 genotypes (odds ratio [OR] 6. 9; 95% confidence interval [CI] 2.5-19.0). This association, which was present whatever the MEFV genotype, was extremely marked in patients homozygous for M694V (11/11). The risk for male patients of developing renal amyloidosis was fourfold higher than that for female patients (OR=4.0; 95% CI=1.5-10.8). This association, particularly marked in patients who were not homozygous for M694V (34.0% vs. 11.6%), was independent of SAA1-allelic variations. Polymorphisms in the SAA2 or APOE gene did not appear to influence susceptibility to renal amyloidosis. Overall, these data, which provide new insights into the pathophysiology of FMF, demonstrate that susceptibility to renal amyloidosis in this Mendelian disorder is influenced by at least two MEFV-independent factors of genetic origin-SAA1 and sex-that act independently of each other.

Species-specific alternative splice mimicry at the growth hormone receptor locus revealed by the lineage of retroelements during primate evolution.

In humans, growth hormone receptor (GHR) transcripts exist in two isoforms differing by the retention (GHRfl) or exclusion (GHRd3) of exon 3, whereas in mice GHRfl is solely expressed. This species-specific expression pattern is believed to result from an alternative splice event that, on the basis of conflicting data obtained in humans, has been considered to be tissue-, developmentally, and/or individual-specific. To decipher the molecular basis of this unusual trait, we isolated a 6.8-kilobase fragment spanning exon 3 from individuals expressing GHRfl. Sequence analysis revealed the existence of two 99% identical retroelements flanking this exon. Unexpectedly, individuals expressing GHRd3 displayed a 2.7-kilobase deletion involving exon 3, which most likely results from an ancestral homologous recombination between the two retroelements. The lineage of these retroelements during primate evolution revealed the species specificity of the GHRd3 allele. These findings led us to propose a model underlying the existence of the sole GHRfl allele in most species. Such a retrovirus-mediated alternative splice mimicry, which clears up several as yet unexplained phenomena (i.e. the above-mentioned expression data, the Mendelian inheritance of GHR expression patterns, and the deletion of nonconsecutive exons in growth hormone resistant patients), represents a novel physiological mechanism accounting for protein diversity between and within species.

A new c-kit mutation in a case of aggressive mast cell disease.

Systemic mast cell disease (SMCD) is a disorder characterized by a mast cell proliferation in various tissues. Mast cells express the c-kit proto-oncogene. A few cases of c-kit mutations have been described in SMCD. We report an aggressive SMCD in a patient who presented with a bone marrow infiltration by abnormal mast cells. Molecular studies of mast cell DNA and RNA revealed a new c-kit heterozygous mutation (Asp820Gly). This mutation leads to a drastic amino-acid change and is located close to the highly oncogenic Asp816Val. These findings suggest that the Asp820Gly has a potential role in c-kit activation.

An exon-skipping mutation in the btk gene of a patient with X-linked agammaglobulinemia and isolated growth hormone deficiency.

X-linked agammaglobulinemia (XLA) is an inherited immunodeficiency disease associated with a block in differentiation from pre-B to B cells. The XLA gene encodes a 659 amino acids cytoplasmic protein tyrosine kinase named btk (Bruton's tyrosine kinase). The few btk gene alterations so far reported in XLA patients are heterogenous and distributed in all domains of the btk protein. They appear to be responsible for a range of B cell immunodeficiency disorders of variable severity. Rare families in which XLA is inherited together with isolated growth hormone deficiency (IGHD) have been reported. Genetic analysis has shown that this disease association maps to the same region of the X chromosome as XLA, but whether the two phenotypes are caused by a common or different developmental or biochemical mechanism is unknown. We have analyzed the btk gene of a patient with XLA and IGHD. RT-PCR analysis of btk transcripts, sequencing data obtained from cDNA and genomic DNA and in vitro splicing assays showed that an intronic point mutation (1882 + 5G-->A) is responsible for skipping of an exon located in the tyrosine kinase domain. This exon-skipping event results in a frameshift leading to a premature stop codon 14 amino acids downstream, and in the loss of the last 61 residues of the carboxy-terminal end of the protein. Although we studied a sporadic case, the results suggest that an alteration of the btk gene might cause this unusual phenotype.

A homozygous insertion-deletion in the type VII collagen gene (COL7A1) in Hallopeau-Siemens dystrophic epidermolysis bullosa.

The Hallopeau-Siemens type of recessive dystrophic epidermolysis bullosa (HS-RDEB) is a life-threatening autosomal disease characterized by loss of dermal-epidermal adherence with abnormal anchoring fibrils (AF). We recently linked HS-RDEB to the type VII collagen gene (COL7A1) which encodes the major component of AF. We describe a patient who is homozygous for an insertion-deletion in the FN-4A domain of the COL7A1 gene. This defect causes a frameshift mutation which leads to a premature stop codon in the FN-5A domain, resulting in a marked diminution in mutated mRNA levels, with no detectable type VII collagen polypeptide in the patient. Our data suggest strongly that this null allele prevents normal anchoring fibril formation in homozygotes and is the underlying cause of HS-RDEB in this patient.

Spectrum of growth hormone receptor mutations and associated haplotypes in Laron syndrome.

Laron syndrome is a rare autosomal recessive disorder characterized by resistance to growth hormone (GH). In 10 patients of different ethnic origins, we have analyzed all the GH receptor (GHR)-coding exons along with their splice junctions and 6 intragenic polymorphic sites defining several GHR gene haplotypes. This allowed us to identify the mutations in the 20 chromosomes studied and to describe a new GHR haplotype. Eleven different mutations associated with various GHR haplotypes were observed; they included 3 nonsense mutations, 3 splice defects and 5 missense mutations. Of the 11 mutations, 8 were novel. All the mutations involved the exoplasmic domain of the receptor and all the missense mutations were clustered in a short polypeptide segment. Most of the missense mutations affected residues conserved among GHRs from different species and the related molecules that belong to the cytokine receptor superfamily. Adding to the 5 mutations so far described, these findings illustrate the allelic heterogeneity of this syndrome and document the independent origin of the molecular defects, all features of clinical relevance for genetic counselling.

Frequent detection of minimal residual disease by use of the polymerase chain reaction in long-term survivors after bone marrow transplantation for chronic myeloid leukemia.

The polymerase chain reaction (PCR) allows the detection of minimal amounts of nucleic sequences and has been successfully used to test for the chronic myeloid leukemia-specific bcr/abl transcripts. We studied blood samples from 17 patients who had undergone allogeneic bone marrow transplantation for CML, using a modified polymerase chain reaction-based assay for the detection of leukemic mRNA. This nested PCR technique was found to be highly sensitive, detecting the chimeric bcr/abl transcript in 16 of 17 patients including several long-term survivors. Cytogenetic techniques failed to detect Ph mitoses. The clinical significance of the persisting bcr/abl transcript for long periods following BMT is poorly understood and remains to be elucidated by further studies.