Homozygous PKP2 deletion associated with neonatal left ventricle noncompaction.

Left ventricular noncompaction cardiomyopathy (LVNC) is a clinically heterogeneous disorder characterized by a trabecular meshwork and deep intertrabecular myocardial recesses that communicate with the left ventricular cavity. Several genetic causes of LVNC have been reported, with variable modes of inheritance, including autosomal dominant and X-linked inheritance, but relatively few responsible genes have been identified. A NGS workflow, based on a panel of 95 genes developed for sequencing most prevalent sudden cardiac death-causing genes, was used to make a rapid and costless molecular diagnosis in two siblings with a severe noncompaction cardiomyopathy starting prenatally and leading to rapid cardiac failure. For the first time, a total homozygous PKP2 deletion was identified. This molecular defect was further confirmed by MLPA and array-comparative genomic hybridization (CGH). Heterozygous PKP2 mutations are usually reported in a significant proportion of Arrhythmogenic Right Ventricular Cardiomyopathy cases. Our results show, for the first time, the involvement of PKP2 in severe cardiomyopathy with ventricular non compaction.

Xq28 duplication including MECP2 in six unreported affected females: what can we learn for diagnosis and genetic counselling?

Duplication of the Xq28 region, involving MECP2 (dupMECP2), has been primarily described in males with severe developmental delay, spasticity, epilepsy, stereotyped movements and recurrent infections. Carrier mothers are usually asymptomatic with an extremely skewed X chromosome inactivation (XCI) pattern. We report a series of six novel symptomatic females carrying a de novo interstitial dupMECP2, and review the 14 symptomatic females reported to date, with the aim to further delineate their phenotype and give clues for genetic counselling. One patient was adopted and among the other 19 patients, seven (37%) had inherited their duplication from their mother, including three mildly (XCI: 70/30, 63/37, 100/0 in blood and random in saliva), one moderately (XCI: random) and three severely (XCI: uninformative and 88/12) affected patients. After combining our data with data from the literature, we could not show a correlation between XCI in the blood or duplication size and the severity of the phenotype, or explain the presence of a phenotype in these females. These findings confirm that an abnormal phenotype, even severe, can be a rare event in females born to asymptomatic carrier mothers, making genetic counselling difficult in couples at risk in terms of prognosis, in particular in prenatal cases.

[Genetic diffuse cystic lung disease in adults]. / Maladies kystiques pulmonaires de l'adulte d'origine génétique.

Multiple cystic lung diseases comprise a wide range of various diseases, some of them of genetic origin. Lymphangioleiomyomatosis (LAM) is a disease occurring almost exclusively in women, sporadically or in association with tuberous sclerosis complex (TSC). Patients with LAM present with lymphatic complications, renal angiomyolipomas and cystic lung disease responsible for spontaneous pneumothoraces and progressive respiratory insufficiency. TSC and LAM have been ascribed to mutations in TSC1 or TSC2 genes. Patients with TSC are variably affected by cutaneous, cognitive and neuropsychiatric manifestations, epilepsy, cerebral and renal tumors, usually of benign nature. Birt-Hogg-Dubé syndrome is caused by mutations in FLCN encoding folliculin. This syndrome includes lung cysts of basal predominance, cutaneous fibrofolliculomas and various renal tumors. The main complications are spontaneous pneumothoraces and renal tumors requiring systematic screening. The mammalian target of rapamycin (mTOR) pathway is involved in the pathophysiology of TSC, sporadic LAM and Birt-Hogg-Dubé syndrome. MTOR inhibitors are used in LAM and in TSC while Birt-Hogg-Dubé syndrome does not progress towards chronic respiratory failure. Future challenges in these often under-recognized diseases include the need to reduce the delay to diagnosis, and to develop potentially curative treatments. In France, physicians can seek help from the network of reference centers for the diagnosis and management of rare pulmonary diseases.

Genetic variation in TGFB1 gene and risk of idiopathic recurrent pregnancy loss.

Transforming growth factor ß1 plays a significant role in pregnancy outcome. We investigated the association of TGFB1 exon 1 (rs1800471, rs1800470) and promoter region (rs1800469, rs1800468) polymorphisms with recurrent pregnancy loss (RPL) in 675 Tunisian women: 304 women with a history of three consecutive pregnancy losses of unknown etiology with the same partner and 371 age-matched multiparous control women. TGFB1 genotyping was done by TaqMan assays. Higher minor allele frequency for rs1800471 (P< 0.001), but not for rs1800470, rs1800469 or rs1800468 was found in RPL cases compared with controls. A significant difference in the distribution of rs1800471 genotypes was seen between the RPL cases and control women, irrespective of the genetic model used. Increased RPL risk was seen with rs1800471 allele C in the heterozygous state and to a greater degree in the homozygous state, thus establishing a dose-dependent effect. Haploview analysis revealed differential linkage disequilibrium between the TGFB1 single-nucleotide polymorphisms analyzed. TGFB1 haplotype analysis identified eight common haplotypes (rs1800471/rs1800470/rs1800469/rs1800468) with three (GTTG, Pc = 0.02; CCTG, Pc = 0.02 and CTCG, Pc = 0.02) positively associated with RPL and one (GCCG, Pc = 0.009) negatively associated with RPL. This study provides the first evidence that the TGFB1 genotype may influence RPL.

Identification of specific vascular endothelial growth factor susceptible and protective haplotypes associated with recurrent spontaneous miscarriages.

BACKGROUND: We investigated the association of vascular endothelial growth factor (VEGF) gene polymorphism with recurrent spontaneous miscarriage (RSM). METHODS: VEGF -2578C/A, -1154G/A, -634G/C, +936C/T single nucleotide polymorphisms (SNPs) were assessed in 304 RSM patients, and 371 age-and body mass index-matched control subjects using real-time PCR. RESULTS: Higher minor allele frequency of -1154G/A (P < 0.001) and +936C/T (P < 0.001), but not -2578C/A (P = 0.55) or -634G/C (P = 0.87) SNPs, were seen in patients. Significant differences in the distribution of -1154G/A (P = 0.006) and +936C/T (P = 0.015), but not -2578C/A (P = 0.473) or -634G/C (P = 1.000) genotypes, were seen in cases compared with control women. Of the possible 16 VEGF haplotypes, 9 were found to be common, and were included. A significantly lower frequency of C G C C (P = 0.008), and A G G C (P < 0.001) haplotypes, and a higher frequency C G C T (P = 0.020), and C G T (P = 0.004) haplotypes were seen in patients. CONCLUSIONS: These results strongly support that VEGF polymorphisms, in particular-1154G/A and +936C/T, are significantly associated with RSM. Our results confirm, in the largest sample to date, previous works in other populations on VEGF polymorphism in RSM.

Tuberous Sclerosis Complex: Genetic counselling and perinatal follow-up.

Tuberous sclerosis is an autosomal dominant disorder almost fully penetrant with highly variable expression. Most cases are de novo and this diagnosis is sometimes considered during prenatal life in case of cardiac tumor, unique or multiple. The couple should be referred to a specialized tertiary prenatal care center for expertise and information. Fetal molecular testing of the two genes TSC1 and TSC2 is often informative. Prognosis determination for Tuberous Sclerosis remains a difficult task. Cardiac tumors can be sometimes worrying but only a minority will have a pejorative issue and most cases are asymptomatic without any therapeutic intervention needed. Only few cases need surgical or medical treatment. Patients with Tuberous Sclerosis can develop skin, eye, kidney or lung lesions later on, but they are either of limited consequence or treatable. The crux of the matter is the neurological involvement with frequent intellectual deficiency and epilepsy that can be drug-resistant. The absence of lesion on fetal brain MRI is not predictive of any prognosis and does not rule out Tuberous Sclerosis. De novo TSC2 mutation is a negative prognosis factor and conversely, an inherited TSC1 mutation is a more favorable one, but with a severe issue still possible. Facing this cautious prognosis, some couple may opt for termination of pregnancy while others decide to pursue it. It is then fundamental to set cardiac and neurological regular follow-up for these newborns. © 2022 French Society of Pediatrics. Published by Elsevier Masson SAS. All rights reserved.

The Renpenning syndrome spectrum: new clinical insights supported by 13 new PQBP1-mutated males.

Since the first reports of polyglutamine-binding protein 1 (PQBP1) mutations in Renpenning syndrome and related disorders, the spectrum of PQBP1-linked clinical manifestations has been outlined from rare published case reports. The phenotypic description is often obtained from medical archives, and therefore, heterogeneous. Moreover, some aspects such as brain imaging or cognitive and behavioral functioning are rarely described. In this study, 13 PQBP1-mutated French patients were subjected to a standardized clinical, cognitive and behavioral assessment. Physical measurements of their relatives were also collected. We report on a recognizable clinical and radiological phenotype. All patients presented with microcephaly, leanness and mild short stature, relative to familial measurements. Three new clinical features are described: upper back progressive muscular atrophy, metacarpophalangeal ankylosis of the thumb and velar dysfunction. The specific facial dysmorphic features included at least four of the following signs: long triangular face, large ridged nose, half-depilated eyebrows, dysplastic or protruding ears and rough slightly sparse hair. An over-aged appearance was noticed in elderly patients. Cortical gyrification was normal based on available magnetic brain imaging of six patients. PQBP1-linked microcephaly (or Renpenning syndrome) is an X-linked mental retardation syndrome, which has clinically recognizable features.

Expanding CEP290 mutational spectrum in ciliopathies.

Ciliopathies are an expanding group of rare conditions characterized by multiorgan involvement, that are caused by mutations in genes encoding for proteins of the primary cilium or its apparatus. Among these genes, CEP290 bears an intriguing allelic spectrum, being commonly mutated in Joubert syndrome and related disorders (JSRD), Meckel syndrome (MKS), Senior-Loken syndrome and isolated Leber congenital amaurosis (LCA). Although these conditions are recessively inherited, in a subset of patients only one CEP290 mutation could be detected. To assess whether genomic rearrangements involving the CEP290 gene could represent a possible mutational mechanism in these cases, exon dosage analysis on genomic DNA was performed in two groups of CEP290 heterozygous patients, including five JSRD/MKS cases and four LCA, respectively. In one JSRD patient, we identified a large heterozygous deletion encompassing CEP290 C-terminus that resulted in marked reduction of mRNA expression. No copy number alterations were identified in the remaining probands. The present work expands the CEP290 genotypic spectrum to include multiexon deletions. Although this mechanism does not appear to be frequent, screening for genomic rearrangements should be considered in patients in whom a single CEP290 mutated allele was identified.

Lethal form of spinocerebellar ataxia type 7 with early onset in childhood.

Progressive cerebellar ataxias are well-known hereditary neurological disorders. Among them, spinocerebellar ataxia type 7 (SCA7) is inherited as an autosomal dominant trait and is ascribed to the expansion of a CAG trinucleotide repeat within the ATXN7 gene. An anticipation phenomenon can occur during paternal transmission and sometimes is responsible for a severe infantile form. The specificity of SCA7 is the retinal involvement with retinitis pigmentosa and cone rod dystrophy. We describe a familial form with two siblings who died of a severe infantile form. Diagnosis was made in their father, who had a recent history of macular atrophy and presented with gait disturbance thereafter. Retrospectively, substantial triplet repeat expansion was confirmed in the two affected infants. These infantile forms are rare and difficult to diagnose in the absence of suggestive family symptoms.

Epistatic interactions with a common hypomorphic RET allele in syndromic Hirschsprung disease.

Hirschsprung disease (HSCR) stands as a model for genetic dissection of complex diseases. In this model, a major gene, RET, is involved in most if not all cases of isolated (i.e., nonsyndromic) HSCR, in conjunction with other autosomal susceptibility loci under a multiplicative model. HSCR susceptibility alleles can harbor either heterozygous coding sequence mutations or, more frequently, a polymorphism within intron 1, leading to a hypomorphic RET allele. On the other hand, about 30% of HSCR are syndromic. Hitherto, the disease causing gene has been identified for eight Mendelian syndromes with HSCR: congenital central hypoventilation (CCHS), Mowat-Wilson (MWS), Bardet-Biedl (BBS), Shah-Waardenburg (WS4), cartilage-hair-hypoplasia (CHH), Smith-Lemli-Opitz (SLO), Goldberg-Sprintzsen (GSS), and hydrocephalus due to congenital stenosis of the aqueduct of sylvius (HSAS). According to the HSCR syndrome, the penetrance of HSCR trait varies from 5 to 70%. Trisomy 21 (T21) also predisposes to HSCR. We were able to collect a series of 393 patients affected by CCHS (n = 173), WS4 (n = 24), BBS (n = 51), MWS (n = 71), T21 (n = 46), and mental retardation (MR) with HSCR (n = 28). For each syndrome, we studied the RET locus in two subgroups of patients; i.e., with or without HSCR. We genotyped the RET locus in 393 patients among whom 195 had HSCR, and compared the distribution of alleles and genotypes within the two groups for each syndrome. RET acts as a modifier gene for the HSCR phenotype in patients with CCHS, BBS, and Down syndrome, but not in patients with MWS and WS4. The frequent, low penetrant, predisposing allele of the RET gene can be regarded as a risk factor for the HSCR phenotype in CCHS, BBS, and Down syndrome, while its role is not significant in MWS and WS4. These data highlight the pivotal role of the RET gene in both isolated and syndromic HSCR.

[The complex phenotype of ARC syndrome: A new case]. / Le phénotype complexe du syndrome ARC : une nouvelle observation.

ARC syndrome (arthrogryposis - renal dysfunction - cholestasis) is a rare lethal multisystemic autosomal recessive disease. A newborn of consanguineous parents of Algerian descent presented cholestatic jaundice, dehydration, and Fanconi syndrome at 10 days of life. The blood smear showed a very characteristic gray appearance of platelets. A homozygous mutation was evidenced in the VPS33B gene. This gene codes for a protein involved in trafficking of intracellular vesicles. The mutation (c.604-2A>G) present in the heterozygous state in the parents affects an invariant base of the splice acceptor site and to our knowledge has not been reported yet. This child died at the age of 3 months. Prenatal diagnosis was offered to the family; another pregnancy was carried to completion and a girl was born without the disease. The combination of cholestasis and proximal tubulopathy should suggest the diagnosis in a newborn with orthopedic problems. A blood smear greatly facilitates diagnosis.

Combined genetic approaches yield a 48% diagnostic rate in a large cohort of French hearing-impaired patients.

Hearing loss is the most common sensory disorder and because of its high genetic heterogeneity, implementation of Massively Parallel Sequencing (MPS) in diagnostic laboratories is greatly improving the possibilities of offering optimal care to patients. We present the results of a two-year period of molecular diagnosis that included 207 French families referred for non-syndromic hearing loss. Our multi-step strategy involved (i) DFNB1 locus analysis, (ii) MPS of 74 genes, and (iii) additional approaches including Copy Number Variations, in silico analyses, minigene studies coupled when appropriate with complete gene sequencing, and a specific assay for STRC. This comprehensive screening yielded an overall diagnostic rate of 48%, equally distributed between DFNB1 (24%) and the other genes (24%). Pathogenic genotypes were identified in 19 different genes, with a high prevalence of GJB2, STRC, MYO15A, OTOF, TMC1, MYO7A and USH2A. Involvement of an Usher gene was reported in 16% of the genotyped cohort. Four de novo variants were identified. This study highlights the need to develop several molecular approaches for efficient molecular diagnosis of hearing loss, as this is crucial for genetic counselling, audiological rehabilitation and the detection of syndromic forms.

l-folinic acid versus d,l-folinic acid in rescue of high-dose methotrexate therapy in children.

BACKGROUND: At this time, folinic acid (FA) is commercially available as the racemic mixture d,l-FA, whose biological activity is supported by natural l-FA. The administration of d,l-FA results in the accumulation of d-FA in plasma relative to the active l-FA form; in vitro studies have shown that d-FA can compete with the polyglutamation of methotrexate (MTX). PURPOSE: Our purpose was to compare, on a pharmacokinetic, biological, and clinical basis, the racemic mixture d,l-FA with the pure l-FA in rescue of high-dose MTX therapy (5 g/m2) in children with acute lymphocytic leukemia (ALL). METHODS: Eighteen children with ALL were entered in this trial, which was planned with a crossover design. Four cycles of MTX were administered to each patient, and rescue was achieved orally every 6 hours at a dose of 12 mg/m2 for d,l-FA and 6 mg/m2 for pure l-FA. The d,l-FA and l-FA rescues were alternated from one cycle to the next. d-FA, l-FA, and the active metabolite 5-methyltetrahydrofolate (5-MTHF) were measured in plasma using a stereospecific high-performance liquid chromatography assay. RESULTS: Considering total active folate levels (l-FA + 5-MTHF), mean residual concentrations were similar for rescue by d,l-FA and l-FA, after two and six intakes, respectively: 92 and 186 nM for d,l-FA rescue versus 100 and 184 nM for l-FA rescue. Intra-individual comparison of total active folates (l-FA + 5-MTHF) did not show any significant difference between d,l-FA rescue and l-FA rescue. After administration of d,l-FA, the accumulation of d-FA in plasma was confirmed. For both types of FA rescue, MTX terminal half-lives were identical (average value, 13.9 hours). Considering each type of toxic effect (hematologic, hepatic, renal, and digestive), there was no significant difference in the proportion of toxic cycles following l-FA rescue or d,l-FA rescue. CONCLUSION: The administration of the pure l-FA, compared with the administration of the racemic mixture, results in comparable blood profiles of active folates and MTX, leads to equivalent treatment tolerance, and avoids the plasma accumulation of d-FA.

Enhancement of the herpes simplex virus thymidine kinase/ganciclovir bystander effect and its antitumor efficacy in vivo by pharmacologic manipulation of gap junctions.

Apigenin, a flavinoid, and lovastatin, an HMG-CoA reductase inhibitor, upregulated gap junction (GJ) function and dye transfer in tumors expressing GJ and were inactive in the GJ-negative tumor line N2a. N2a cells transfected with the connexin 43 gene showed restored cell-to-cell dye transfer, which could then be improved nearly fourfold by addition of apigenin. To test the drugs in HSV thymidine kinase/ganciclovir (HSV-tk/GCV) tumor killing, mixtures of 90% wild-type (WT) with 10% HSV-tk gene-modified MCA38 adenocarcinoma cells were exposed in vitro to GCV +/- apigenin or lovastatin. A significant bystander effect (BSE) was seen following GCV treatment alone, while neither apigenin or lovastatin alone had any effect on the recovery of viable tumor colonies. However, GCV-treated cultures also exposed to apigenin or lovastatin showed an increased BSE and reduced tumor cell recovery. Thirty percent of mice bearing tumors from the same mixture of 90% WT and 10% HSV-tk MCA38 cells treated with GCV alone became tumor free. Tumor-bearing mice given only two or three injections of lovastatin or apigenin during GCV treatment had a doubling of the antitumor response rate, with 60-70% of the mice achieving complete remission. These results support the hypothesis that the transfer of phosphorylated GCV from HSV-tk gene-expressing cells to neighboring WT tumor cells is a major component of the BSE and that pharmacological manipulation of GJ function with lovastatin or apigenin can result in striking improvement in the antitumor response in mice with tumors modified to contain as few as 10% HSV-tk cells.

Neutrophil studies in psoriatics: in vivo migration, phagocytosis and bacterial killing.

A reproducible method for sequential study of migration out of human skin, phagocytosis and bactericidal activity of neutrophils is described. Untreated psoriatics exhibit an early increase of chemotactic activity (0-8 hr, p less than 0.02) and subsequently a strong inhibition of chemotaxis (8-24 hr, p less than 0.01), a slight decrease of phagocytosis and a decrease in bactericidal activity (20 min, p less than 0.02; 30 min, p less than 0.003); 60 min, p less than 0.001; 120 min, p less than 0.001) as compared with controls. After clearing of skin lesions, the early increased chemotactic activity returned to normal values but the subsequent chemotactic inhibition remains as strong as before treatment. Phagocytosis increased to normal values (p less than 0.02) and bactericidal activity also increased but remained significantly low. These abnormalities were more evident in migrating than in circulating neutrophils, underlining the sensitivity of the described method.

The contractility of fibroblasts in a collagen lattice is reduced by corticosteroids.

Normal human and rat skin fibroblasts, incorporated into a three-dimensional collagen lattice, were assayed for their ability to contract collagen fibrils. This capacity is inhibited in a dose-dependent fashion by both dexamethasone and hydrocortisone. The inhibition is reversed when the corticosteroids are removed from the culture medium, but while the effect of hydrocortisone is almost entirely reversible, that of dexamethasone is not.

Diaminobenzidine cytochemistry in unfixed human epidermis: a marker for epidermal differentiation and for mitochondria.

Intubation of unfixed and unfrozen slices of skin in diaminobenzidine allows visualization of a peroxidatic activity in perinuclear envelope of suprabasal keratinocytes undergoing orthokeratotic differentiation. Basal keratinocytes and melanocytes are always negative. This enzyme is absent in mucous and parakeratotic (psoriatic) differentiation. Mitochondria are also strongly stained by this technique and it was shown that the number of epidermal mitochondria is greatly increased in psoriatic lesions.

Endogenous peroxidases in normal human dermis: a marker of fibroblast differentiation.

Incubation of unfixed and unfrozen slices of normal human skin allows visualization of a peroxidase activity associated with the perinuclear envelope and with the endoplasmic reticulum of resident dermal macrophages, dermal mastocytes, and also of some dermal fibroblasts. No peroxidase activity can be detected in fibroblasts cultivated in monolayer, while 80% of fibroblasts in an "in vitro" collagen lattice, called a dermal equivalent, express a peroxidase activity in the perinuclear envelope and the endoplasmic reticulum. Hence expression of this peroxidase activity in normal human skin fibroblasts serves as a marker of fibroblast differentiation and seems to depend on an interaction to fibroblast with the elements of a three-dimensional matrix.

Identification of fifteen novel mutations in the tissue-nonspecific alkaline phosphatase (TNSALP) gene in European patients with severe hypophosphatasia.

Hypophosphatasia is an inherited disorder characterised by defective bone mineralisation and deficiency of serum and tissue liver/bone/kidney alkaline phosphatase (L/B/K ALP) activity. We report the characterisation of tissue-nonspecific alkaline phosphatase (TNSALP) gene mutations in a series of 13 European families affected by perinatal, infantile or childhood hypophosphatasia. Eighteen distinct mutations were found, only three of which had been reported previously in North American and Japanese populations. Most of the 15 new mutations were missense mutations, but we also found two mutations affecting donor splice sites and a nonsense mutation. A missense mutation in the last codon of the putative signal peptide probably affects the final maturation of the protein. Despite extensive sequencing of the gene and its promotor region, only one mutation was identified in two cases, one of which was compatible with a possible dominant effect of certain mutations and the putative role of polymorphisms of the TNSALP gene. In 12 of the 13 tested families, genetic diagnosis was possible by characterisation of the mutations or by use of polymorphisms as genetic markers. Hypophosphatasia diagnosis was assigned in two families where clinical, laboratory and radiographic data were unclear and prenatal diagnosis was performed in one case. The results also show that severe hypophosphatasia is due to a very large spectrum of mutations in European populations with no prevalent mutation and that genetic diagnosis of the disease must be performed by extensive analysis of the gene.