Catherine Dodé

Locus assignment of human a globin mutations by selective amplification and direct sequencing

Summary We describe a simple approach for molecular characterization and locus assignment of structural mutants by direct sequencing of enzymatically amplified DNA selective to α1 and α2 globin gene regions. Nucleotide substitution of two structural variants (Stanleyville II α278Lys and J Mexico α254Glu) were determined and their encoding loci were specified. The amplified segment encompasses sequences upstream of the CAAT box to downstream of the Poly(A) addition signal. Hence all of the α globin structural variants and most of the nondeletion α thalassaemic mutants should be characterizable by this approach.

Rapid analysis of ‐α3.7 thalassaemia and αααanti 3.7 triplication by enzymatic amplification analysis

Summary In this report we describe a PCR‐based method for the diagnosis of the most common form of α thalassaemia, the –α3.7 deletion which occurs throughout all tropical and subtropical regions of the world. The same procedure also identifies the reciprocal recombinant chromosome (αααanti 3.7). Restriction mapping of the PCR products has enabled us to distinguish between the type I (–α3.7I), type II (–α3.7II) and type III (–α3.7III) deletions. This strategy will be very useful in screening programmes of α thalassaemia occurring on its own or in association with β thalassaemia and sickle cell disease.

A Comparative Phenotypic Study of Kallmann Syndrome Patients Carrying Monoallelic and Biallelic Mutations in the Prokineticin 2 or Prokineticin Receptor 2 Genes

CONTEXTnBoth biallelic and monoallelic mutations in PROK2 or PROKR2 have been found in Kallmann syndrome (KS).nnnOBJECTIVEnThe objective of the study was to compare the phenotypes of KS patients harboring monoallelic and biallelic mutations in these genes.nnnDESIGN AND PATIENTSnWe studied clinical and endocrine features that reflect the functioning of the pituitary-gonadal axis, and the nonreproductive phenotype, in 55 adult KS patients (42 men and 13 women), of whom 41 had monoallelic mutations and 14 biallelic mutations in PROK2 or PROKR2.nnnRESULTSnBiallelic mutations were associated with more frequent cryptorchidism (70% vs. 34%, P < 0.05) and microphallus (90% vs. 28%, P < 0.001) and lower mean testicular volume (1.2 +/- 0.4 vs. 4.5 +/- 6.0 ml; P < 0.01) in male patients. Likewise, the testosterone level as well as the basal FSH level and peak LH level under GnRH-stimulation were lower in males with biallelic mutations (0.2 +/- 0.1 vs. 0.7 +/- 0.8 ng/ml; P = 0.05, 0.3 +/- 0.1 vs. 1.8 +/- 3.0 IU/liter; P < 0.05, and 0.8 +/- 0.8 vs. 5.2 +/- 5.5 IU/liter; P < 0.05, respectively). Nonreproductive, nonolfactory anomalies were rare in both sexes and were never found in patients with biallelic mutations. The mean body mass index of the patients (23.9 +/- 4.2 kg/m(2) in males and 26.3 +/- 6.6 kg/m(2) in females) did not differ significantly from that of gender-, age-, and treatment-matched KS individuals who did not carry a mutation in PROK2 or PROKR2. Finally, circadian cortisol levels evaluated in five patients, including one with biallelic PROKR2 mutations, were normal in all cases.nnnCONCLUSIONnMale patients carrying biallelic mutations in PROK2 or PROKR2 have a less variable and on average a more severe reproductive phenotype than patients carrying monoallelic mutations in these genes. Nonreproductive, nonolfactory clinical anomalies associated with KS seem to be restricted to patients with monoallelic mutations.

Loss-of-Function Mutations in SOX10 Cause Kallmann Syndrome with Deafness

Transcription factor SOX10 plays a role in the maintenance of progenitor cell multipotency, lineage specification, and cell differentiation and is a major actor in the development of the neural crest. It has been implicated in Waardenburg syndrome (WS), a rare disorder characterized by the association between pigmentation abnormalities and deafness, but SOX10 mutations cause a variable phenotype that spreads over the initial limits of the syndrome definition. On the basis of recent findings of olfactory-bulb agenesis in WS individuals, we suspected SOX10 was also involved in Kallmann syndrome (KS). KS is defined by the association between anosmia and hypogonadotropic hypogonadism due to incomplete migration of neuroendocrine gonadotropin-releasing hormone (GnRH) cells along the olfactory, vomeronasal, and terminal nerves. Mutations in any of the nine genes identified to date account for only 30% of the KS cases. KS can be either isolated or associated with a variety of other symptoms, including deafness. This study reports SOX10 loss-of-function mutations in approximately one-third of KS individuals with deafness, indicating a substantial involvement in this clinical condition. Study of SOX10-null mutant mice revealed a developmental role of SOX10 in a subpopulation of glial cells called olfactory ensheathing cells. These mice indeed showed an almost complete absence of these cells along the olfactory nerve pathway, as well as defasciculation and misrouting of the nerve fibers, impaired migration of GnRH cells, and disorganization of the olfactory nerve layer of the olfactory bulbs.

PROK2/PROKR2 Signaling and Kallmann Syndrome

Kallmann syndrome (KS) is a developmental disease that associates hypogonadism and a deficiency of the sense of smell. The reproductive phenotype of KS results from the primary interruption of the olfactory, vomeronasal, and terminal nerve fibers in the frontonasal region, which in turn disrupts the embryonic migration of neuroendocrine gonadotropin-releasing hormone (GnRH) synthesizing cells from the nose to the brain. This is a highly heterogeneous genetic disease, and mutations in any of the nine genes identified so far have been found in approximately 30% of the KS patients. PROKR2 and PROK2, which encode the G protein-coupled prokineticin receptor-2 and its ligand prokineticin-2, respectively, are two of these genes. Homozygous knockout mice for the orthologous genes exhibit a phenotype reminiscent of the KS features, but biallelic mutations in PROKR2 or PROK2 (autosomal recessive mode of disease transmission) have been found only in a minority of the patients, whereas most patients carrying mutations in these genes are heterozygotes. The mutations, mainly missense mutations, have deleterious effects on PROKR2 signaling in transfected cells, ranging from defective cell surface-targeting of the receptor to defective coupling to G proteins or impaired receptor-ligand interaction, but the same mutations have also been found in apparently unaffected individuals, which suggests a digenic/oligogenic mode of inheritance of the disease in heterozygous patients. This non-Mendelian mode of inheritance has so far been confirmed only in a few patients. However, it may account for the unusually high proportion of KS sporadic cases compared to familial cases.

Kallmann syndrome caused by mutations in the PROK2 and PROKR2 genes: pathophysiology and genotype-phenotype correlations.

Mutations in the prokineticin 2 peptide (PROK2) and its seven-transmembrane domain type 2 receptor PROKR2 are newly identified molecular culprits in autosomal Kallmann syndrome (KS). Prok2 and prokr2 gene knockout mice both have agenesis or hypoplasia of the olfactory bulbs, associated with hypogonadotropic hypogonadism linked to abnormal GnRH neuron migration. Prok2-/- and prokr2-/- mice are the first murine models of this human disease. KS patients of both sexes have a variety of point mutations, missense mutations, frameshifts and nonsense mutations in the PROK2 and PROKR2 genes that lead to a loss of peptide or receptor function. When only one allele is affected, penetrance of the two main clinical features of KS may be incomplete: subjects with only one mutant allele may have (1) no symptoms, with normal olfaction and complete pubertal development, (2) congenital isolated (idiopathic) hypogonadotropic hypogonadism (IHH) but normal olfaction, (3) anosmia/hyposmia but normal pubertal development and gonadal function or (4) the two cardinal clinical KS signs, anosmia and IHH. These phenotypic dissociations can be seen in family members with the same PROK2/PROKR2 mutations. By contrast, patients with two mutant alleles almost always have the cardinal signs of KS. Even when monoallelic PROK2/PROKR2 mutations are associated with full-blown KS, the reproductive phenotype in males is less severe than in KS associated with biallelic mutations, evidenced by significantly lower frequency of cryptorchidism and micropenis, greater testicular volume, and higher serum levels of LH, FSH and testosterone. Moreover, at least some of these monoallelic cases are in fact digenic, in that they also carry mutations of other KS/IHH genes. Overall, these observations point towards a combination of mendelian autosomal recessive transmission, with more complex oligogenic transmission. Patients with this genetic form of KS have been reported to have a possible increased prevalence of obesity and sleep disorders, which may be related to the role of PROK2 and PROKR2 in food intake and circadian rhythms. However, diurnal variation of serum cortisol levels appears to be physiologically maintained.

Isolation and characterization of the vitamin K dependent domain of human prothrombin

Abstract Chymotryptic cleavage of human prothrombin produces two fragments, prothrombin (des 1–44) previously characterized and peptide 1–41. This peptide has been purified by barium citrate adsorption and Sephadex G100 chromatography. It contains the 10 γ-carboxyglutamic residues of prothrombin. Added to a prothrombin activation mixture containing FXa, phospholipid and Ca ++ , peptide 1–41 inhibits thrombin generation with the same potency as prothrombin fragment 1. Ca ++ produces a 20 % quenching of the intrinsic fluorescence of the peptide. So do Mn ++ and Mg ++ although to a lesser extent. Phospholipid enhances the Ca ++ induced quenching by a factor of 1.7 and shifts the midpoint of the transition from 0.34 to 0.46 mM Ca ++ . The major difference with titration curves obtained with prothrombin F1 is the absence of cooperativity. Hence peptide 1–44 has retained some of the prothrombin properties to interact with Ca ++ and phospholipid and represents the vitamin K dependent domain of the molecule. The presence of the remaining part of F1 (residues 44–155) however is necessary for the expression of cooperativity.

Fièvres intermittentes héréditaires

Resume En dehors de la fievre mediterraneenne familiale Quatre maladies hereditaires se presentant sous forme d’acces inflammatoires intermittents ont ete reconnues et maintenant bien caracterisees dans leurs aspects cliniques et genetiques. Le chef de file de ce groupe reste la fievre mediterraneenne familiale (FMF), une maladie qui affecte des milliers de personnes originaires du Bassin mediterraneen. Mais la fievre mediterraneenne familiale n’est plus seule au sein des maladies inflammatoires recurrentes hereditaires. Trois autres entites clinico-genetiques maintenant bien definies Il s’agit de la fievre intermittente secondaire a des mutations du recepteur de type 1A du Tumor Necrosis Factor (TNF), de transmission genetique autosomique dominante, le syndrome d’hyperimmunoglobulinemie D, et une derniere entite regroupant le syndrome de Muckle-Wells, l’urticaire familiale au froid et le syndrome CINCA (Chronic Infantile Neurological Cutaneous and Articular) . En pratique Un diagnostic precis de ces affections est crucial, car leur prise en charge et leur traitement sont specifiques.

CHARACTERIZATION OF A PROTEOLYTICALLY MODIFIED FORM OF HUMAN PROTHROMBIN

Abstract Treatment of human prothrombin by α chymotrypsin results in the production of a single homogeneous product with an apparent M.W. of 69 000. This product fails to adsorb on barium citrate. NH2-terminal sequence analysis shows that a single cleavage has occurred after tyrosine 44. The proteolytically modified prothrombin which can therefore be referred to as prothrombin (des 1–44), lacks the whole vitamin K dependent part of the molecule. The region of the peptide chain around tyrosine 44 must be particularly exposed to proteolytic attack and serve as a junction between the vitamin K dependent domain and the other structural domains of prothrombin. In the presence of factor Xa alone, prothrombin (des 1–44) is indistinguishable from normal prothrombin when activation is monitored by the appearance of amidolytic activity on S2238. However, activation of prothrombin (des 1–44) is no longer enhanced by the presence of Ca++ and phospholipid in the activation mixture.

Analysis of crossover type in the α-3·7 haplotype among sickle cell anemia patients from various parts of Africa

SummaryThe frequency of α+-thalassmia has been determined in African populations carrying βs-chromosomes of different origins. All these α+ thalassemias result from a right-ward deletion. Restriction mapping of the α-3·7/haplotype with the enzyme ApI only showed the presence of a type I crossover. RsaI polymorphism at the 5′ end of Zα2 is largely represented in the normal population (gene frequency 23%) but, in our series, never associated with the α-3·7/haplotype.