ARL6IP1 mutation causes congenital insensitivity to pain, acromutilation and spastic paraplegia.

Hereditary sensory and autonomic neuropathies (HSAN) type II are characterized by autosomal recessive inheritance, onset at birth and self-mutilating behavior. Here, we described a new patient with congenital insensitivity to pain, sensory neuropathy, acromutilation, and spastic paraplegia. Whole-exome sequencing showed a homozygous frameshift variant c.[577_580del], p.(Lys193Phefs*37) in ARL6IP1. The protein harbors reticulon-like short hairpin transmembrane domains and has a role in endoplasmic reticulum shaping. The variant causes an additional C-terminus hydrophobic domain which could disrupt its function. ARL6IP1 interacts with atlastin-1 responsible for SPG3A and HSAN type ID. This report highlights the role of ARL6IP1 in the pathophysiology of insensitivity to pain and spastic paraplegia.

Two novel variants of uncertain significance in GP9 associated with Bernard-Soulier syndrome: Are they true mutations?

Bernard-Soulier syndrome (BSS) is an autosomal recessive major thrombocytopathy, the symptoms of which are mainly marked by mucocutaneous bleeding. This rare disease, initially described in the 1970s, is the result of an abnormal formation of the glycoprotein complex Ib-IX-V (GP Ib-IX-V), a platelet receptor of von Willebrand factor. A large number of mutations, sometimes involving the GP9 gene, have been described as possibly responsible for the disease. We report here the case of a BSS patient who presented with persistent thrombocytopenia (31x109/L) and decreased surface expression of GPIb-IX-V on large platelets with anisocytosis. Thorough molecular analyses disclosed two previously unreported GP9 variants, respectively c.230T>A (p.Leu77Gln) and c.255C>A (p.Asn85Lys). Both are likely to modify the conformation of GP-IX interactions with other glycoproteins of the Ib-IX-V complex and thus proper expression of this complex on the membrane of platelets.

Use of a thrombopoietin receptor agonist in von Willebrand disease type 2B (p.V1316M) with severe thrombocytopenia and intracranial hemorrhage.

We present here a 63-year old woman with a long history of immune thrombocytopenia. She was hospitalized for a traumatic intracranial hemorrhage with thrombocytopenia. Following inefficient treatment of four platelet transfusions, immunoglobulins, and corticosteroids, we initiated treatment with a thrombopoietin (TPO) receptor agonist (eltrombopag 25 mg/d) with a good efficacy. Her mother and sister also had chronic thrombocytopenia. Clinical history, hemostasis results, and gene analysis revealed von Willebrand disease (VWD) type 2B with the mutation (c.3946G>A; p.V1316M), which combines a von Willebrand factor defect with severe thrombocytopenia, as well as a thrombocytopathy. The efficacy of TPO receptor agonists appears to counterbalance, at least to some extent, the thrombocytopathy associated with this mutation. As such, the use of TPO receptor agonists could represent an alternative therapeutic approach in cases of VWD type 2B with severe thrombocytopenia.

Platelet function analyser (PFA-100) results and von Willebrand factor deficiency: a 16-year 'real-world' experience.

The platelet function analyser (PFA-100) is a biological tool designed to explore primary haemostasis. This system has thus been widely demonstrated as reliable in detecting von Willebrand factor (VWF) deficiency. However, most studies were based on patients benefitting from regular medical care and accurate diagnosis, and it would seem probable that the results were somewhat optimistic, and do not reflect its performances in 'real-world' situations. We have chosen to study the reliability of PFA-100 for screening VWF ristocetin cofactor (VWF:RCo) deficiency. We retrospectively analysed the results (n = 6431) of 4027 patients referred to our centre between October 1997 and June 2013 and in whom PFA-Epi, PFA-ADP, and VWF:RCo activity had been evaluated. We studied the influence of blood group on the results and the performances of each method in a subgroup of 213 patients with genetically confirmed von Willebrand disease. We have shown that the PFA-100 system, in our experience, constitutes an excellent screening test for detecting VWF:RCo deficiency, whatever the clinical situation, in 'real-world' conditions. The negative predictive value (NPV), the positive predictive value, the sensitivity and the specificity were respectively: 0.98, 0.51, 0.98 and 0.40. When values adjusted for blood group are used, NPV and sensitivity are inferior to those using normal values which have not been adjusted for blood group. We have shown the PFA-100 method to be more efficient in screening for VWF deficiency than the VWF:RCo technique.

Prevalence, biological phenotype and genotype in moderate/mild hemophilia A with discrepancy between one-stage and chromogenic factor VIII activity.

BACKGROUND: In most laboratories, the severity of hemophilia A is assessed by the factor VIII activity (FVIII:C) one-stage assay. However, comparisons of these results with those of two-stage assays can reveal discrepancies and suggest misdiagnosis. PATIENTS/METHODS: In this monocentric study, we measured FVIII:C with two methods (one-stage chronometric and chromogenic assays) in 307 (173 families) patients with moderate/mild hemophilia A. To compare results, we used a chronometric/chromogenic ratio. Discrepancy was defined as a ratio < 0.5 or > 1.5. We studied their putative involvement at known FVIII functional sites, their interspecies conservation status, and their spatial position within the FVIII structure. RESULTS: Thirty-six patients from 17 families exhibited a discrepancy between the two assays: 12 (6.9%) families had a low ratio (< 0.5), and five (2.9%) families had a high ratio (> 1.5). Qualitative deficiency was diagnosed in about 16% of the families. Molecular studies were performed in 15 of these 17 families, resulting in each case in the identification of missense mutations, including three novel mutations. We were further able to propose a pathophysiologic explanation. CONCLUSIONS: In this monocentric study, we have demonstrated a discrepancy between FVIII:C assay results in 10% of families with moderate/mild hemophilia A. The prevalence of 'inverse' discrepancy (i.e. low chronometric/chromogenic ratio) is high as compared with previous reports. We suggest that both FVIII:C assays are recommended in patients with moderate/mild hemophilia A for a complete biological phenotype. This could also improve our knowledge of the FVIII structure-function relationships.

The very low penetrance of cystic fibrosis for the R117H mutation: a reappraisal for genetic counselling and newborn screening.

BACKGROUND: Cystic fibrosis (CF) is caused by compound heterozygosity or homozygosity of CF transmembrane conductance regulator gene (CFTR) mutations. Phenotypic variability associated with certain mutations makes genetic counselling difficult, notably for R117H, whose disease phenotype varies from asymptomatic to classical CF. The high frequency of R117H observed in CF newborn screening has also introduced diagnostic dilemmas. The aim of this study was to evaluate the disease penetrance for R117H in order to improve clinical practice. METHODS: The phenotypes in all individuals identified in France as compound heterozygous for R117H and F508del, the most frequent CF mutation, were described. The allelic prevalences of R117H (p(R117H)), on either intron 8 T5 or T7 background, and F508del (p(F508del)) were determined in the French population, to permit an evaluation of the penetrance of CF for the [R117H]+[F508del] genotype. RESULTS: Clinical details were documented for 184 [R117H]+[F508del] individuals, including 72 newborns. The disease phenotype was predominantly mild; one child had classical CF, and three adults' severe pulmonary symptoms. In 5245 healthy adults, p(F508del) was 1.06%, p(R117H;T7) 0.27% and p(R117H;T5)<0.01%. The theoretical number of [R117H;T7]+[F508del] individuals in the French population was estimated at 3650, whereas only 112 were known with CF related symptoms (3.1%). The penetrance of classical CF for [R117H;T7]+[F508del] was estimated at 0.03% and that of severe CF in adulthood at 0.06%. CONCLUSIONS: These results suggest that R117H should be withdrawn from CF mutation panels used for screening programmes. The real impact of so-called disease mutations should be assessed before including them in newborn or preconceptional carrier screening programmes.

Penetrance of marked cognitive impairment in older male carriers of the FMR1 gene premutation.

BACKGROUND: Male carriers of the FMR1 premutation are at risk of developing the fragile X-associated tremor/ataxia syndrome (FXTAS), a newly recognised and largely under-diagnosed late onset neurodegenerative disorder. Patients affected with FXTAS primarily present with cerebellar ataxia and intention tremor. Cognitive decline has also been associated with the premutation, but the lack of data on its penetrance is a growing concern for clinicians who provide genetic counselling. METHODS: The Mattis Dementia Rating Scale (MDRS) was administered in a double blind fashion to 74 men aged 50 years or more recruited from fragile X families (35 premutation carriers and 39 intrafamilial controls) regardless of their clinical manifestation. Based on previous publications, marked cognitive impairment was defined by a score

Mild hemophilia A with factor VIII assay discrepancy: using thrombin generation assay to assess the bleeding phenotype.

INTRODUCTION: In some patients with mild hemophilia A, there are discrepancies between 1-stage (1-st) and 2-stage (2-st) factor VIII (FVIII) clotting assays, and also chromogenic assays for FVIII activity (FVIII:C). We examined whether thrombography could provide a better evaluation of the hemostatic status of these patients. METHODS: Two families with such discrepancies and markedly contrasting clinical histories were studied. Family X had no serious bleedings, in contrast to family Y. Sixty-one moderate/mild hemophiliacs without discrepancy and 15 healthy subjects served as controls. Calibrated automated thrombography was performed with platelet-rich plasma after one freeze-thawing cycle and low tissue factor concentration. RESULTS: The chromogenic FVIII:C levels were higher (0.90 +/- 0.15 and 0.47 +/- 0.13 IU mL(-1)) than the 1-st clotting ones (0.14 +/- 0.05 and 0.10 +/- 0.05 IU mL(-1)) in family X and Y, respectively (P < 0.001). Mean endogenous thrombin potential (ETP) was 1579 +/- 359 nM min(-1) and 1060 +/- 450 for healthy controls and hemophilic controls, respectively. For members of family X, the ETP values were 1188, 1317 and 2277 nM min(-1), whereas for those of family Y they ranged from 447 to 1122 nM min(-1). Two novel missense point mutations were evidenced: p.Ile369Thr in family X and p.Phe2127Ser in family Y. In family X, we postulate that the mutation is responsible for a delayed but non-deleterious FVIII activation. CONCLUSIONS: Our results suggest that the hemostatic phenotype assessed by thrombography may be clinically relevant in moderate/mild hemophilic patients with discrepant FVIII:C results.

Novel SCN5A mutation leading either to isolated cardiac conduction defect or Brugada syndrome in a large French family.

BACKGROUND: The SCN5A gene encoding the human cardiac sodium channel alpha subunit plays a key role in cardiac electrophysiology. Mutations in SCN5A lead to a large spectrum of phenotypes, including long-QT syndrome, Brugada syndrome, and isolated progressive cardiac conduction defect (Lenègre disease). METHODS AND RESULTS: In the present study, we report the identification of a novel single SCN5A missense mutation causing either Brugada syndrome or an isolated cardiac conduction defect in the same family. A G-to-T mutation at position 4372 was identified by direct sequencing and was predicted to change a glycine for an arginine (G1406R) between the DIII-S5 and DIII-S6 domain of the sodium channel protein. Among 45 family members, 13 were carrying the G1406R SCN5A mutation. Four individuals from 2 family collateral branches showed typical Brugada phenotypes, including ST-segment elevation in the right precordial leads and right bundle branch block. One symptomatic patient with the Brugada phenotype required implantation of a cardioverter-defibrillator. Seven individuals from 3 other family collateral branches had isolated cardiac conduction defects but no Brugada phenotype. Three flecainide test were negative. One patient with an isolated cardiac conduction defect had an episode of syncope and required pacemaker implantation. An expression study of the G1406R-mutated SCN5A showed no detectable Na(+) current but normal protein trafficking. CONCLUSIONS: We conclude that the same mutation in the SCN5A gene can lead either to Brugada syndrome or to an isolated cardiac conduction defect. Our findings suggest that modifier gene(s) may influence the phenotypic consequences of a SCN5A mutation.

Dynamic analysis of the QT interval in long QT1 syndrome patients with a normal phenotype.

AIMS: In families with the long QT syndrome penetrance may be low: up to 70% of gene carriers may have a normal QTc interval. These patients require therapy, similar to that in those with longer QTc intervals, but identifying them, using molecular analysis, is difficult to apply on a large scale. A large French family affected by the long QT1 syndrome was followed-up over a 25-year period. In adult males but not in females, the QTc interval normalized after puberty. We aimed to find clinical criteria, based on ambulatory ECG recordings so that we could improve diagnosis in affected members with a normal QTc. METHODS AND RESULTS: Linkage analysis and direct sequencing were an indicator of the long QT1 gene in our family. Reverse transcription-polymerase chain reaction analysis demonstrated abnormal transcripts in lymphocytes from silent gene carriers. The functional profile of mutated protein isoforms was investigated using the patch-clamp technique. Dynamic analysis of ventricular depolarization was conducted using Holter recordings in patients, and in sex- and age-matched controls. Circadian variations of the QTc interval and the QT/RR relationship were assessed. Sensitivity, specificity, and predictive values were evaluated for proposed clinical criteria. We found that dynamic analysis of the QT interval permitted individual diagnosis in mutation carriers even when the QTc interval was normal (adult males). CONCLUSION: Dynamic analysis of the QT interval is of diagnostic value in the long QT1 syndrome in patients with a normal phenotype. Clinical implications include improvement in screening and patient management.

Respiratory epithelial ion transport in patients with disseminated bronchiectasis.

The nosological limits between disseminated bronchiectasis and cystic fibrosis (CF) remain unclear. In patients with isolated congenital bilateral absence of the vas deferens, a forme fruste of the CF disease, a normal baseline nasal transepithelial potential difference (PD) but an impaired response to pharmacological interventions have been reported. The purpose of the present study was to explore ion transport in respiratory epithelium from patients with disseminated bronchiectasis. The PD under both baseline and pharmacological interventions was investigated in 13 healthy subjects, six patients with genetically proven CF and 15 patients with disseminated bronchiectasis as confirmed by computed tomography scan. Baseline PD was similar in the control and bronchiectasis groups but, as expected, was significantly more negative in the CF group. Patients with bronchiectasis responded to pharmacological tests (sequential perfusion with amiloride, chloride-free solution, isoprenaline and uridine triphosphate (UTP) similarly to healthy subjects. In contrast, CF patients exhibited an increased response to amiloride and an impaired response to chloride-free solution and isoprenaline. The data show that patients with disseminated bronchiectasis exhibit normal electrophysiological properties in their nasal epithelium. Nasal transepithelial potential difference including pharmacological tests may appear a valuable diagnostic procedure for cystic fibrosis with disseminated bronchiectasis.

Protease-activated receptor genes are clustered on 5q13.

The serine protease, thrombin, is both a potent agonist for platelet aggregation and a mitogen inducing the proliferation of other cell types. Many cellular responses to thrombin are mediated by a G-protein-coupled thrombin receptor (protease-activated receptor-1, PAR-1). This represents the prototype of a new family of proteolytically cleaved receptors that includes PAR-2 and the recently identified PAR-3. Like PAR-1, PAR-3 is a potential thrombin receptor. Their similar gene structure, mechanism of activation, and colocalization to 5q13 raises the question of a common evolutionary origin and of their belonging to a clustered gene family. Construction of a physical map of the 5q13 region by pulsed-field gel electrophoresis (PFGE) has allowed us to identify six potential CpG islands and to establish a linkage of the PAR genes. Southern blot analysis showed that they were in a cluster on a 560-kb Asc I fragment, in the order PAR-2, PAR-1, and PAR-3. PAR-1 and PAR-2 genes were contained within the identical 240-kb Not I fragment, thus confirming a tight linkage between them. The localization of other CpG islands suggested that more PAR-family genes may be present.

Refined localisation of the voltage-gated chloride channel, CLCN3, to 4q33.

Mutations in ion channels have been shown to be responsible for a variety of neurological and muscular diseases. The voltage-gated chloride channel CLCN3 was recently mapped to chromosomal region 4q32. We are analysing a young female patient with Wolf-Hirschhorn syndrome and chorea associated with an inversion-deletion of chromosome 4 [46XX,inv(4)del(4)(qter-->q33::p15.32-->q33]. Considering that chorea in this patient might be due to the disruption of a gene at either of the 4p15.32 or 4q33 breakpoints, CLCN3 was considered as a candidate gene. We showed by FISH analysis with a CLCN3 YAC that the gene was not broken by the inv-del event, and was therefore an unlikely candidate. Using high resolution techniques, we refined the localisation of CLCN3 to 4q33.

Seeding of YACs over regions 1q41-q42.3 and 11q14.3-q23 with microdissection clones.

We describe the use of pooled, region-specific hybridisation probes to screen high-density replica filters of a human genome YAC library. The probes were derived by microdissection of an approximately 30-Mbp region subtending the translocation breakpoint on a der(1)(1;11)(q42.1;q14.3) chromosome. Of 70 microdissection clones used in pools of 4-10, 47 identified a total of 77 YAC recombinants, representing over 50% of the microdissected region. This strategy can easily be adapted to other poorly mapped subchromosomal regions of the human or other mammalian genomes and will provide a solid framework for detailed contig map constructions.