Expanding the genetic and clinical spectrum of Tatton-Brown-Rahman syndrome in a series of 24 French patients.

BACKGROUND: Tatton-Brown-Rahman syndrome (TBRS; OMIM 615879), also known as DNA methyltransferase 3 alpha (DNMT3A)-overgrowth syndrome (DOS), was first described by Tatton-Brown in 2014. This syndrome is characterised by overgrowth, intellectual disability and distinctive facial features and is the consequence of germline loss-of-function variants in DNMT3A, which encodes a DNA methyltransferase involved in epigenetic regulation. Somatic variants of DNMT3A are frequently observed in haematological malignancies, including acute myeloid leukaemia (AML). To date, 100 individuals with TBRS with de novo germline variants have been described. We aimed to further characterise this disorder clinically and at the molecular level in a nationwide series of 24 French patients and to investigate the correlation between the severity of intellectual disability and the type of variant. METHODS: We collected genetic and medical information from 24 individuals with TBRS using a questionnaire released through the French National AnDDI-Rares Network. RESULTS: Here, we describe the first nationwide French cohort of 24 individuals with germline likely pathogenic/pathogenic variants in DNMT3A, including 17 novel variants. We confirmed that the main phenotypic features were intellectual disability (100% of individuals), distinctive facial features (96%) and overgrowth (87%). We highlighted novel clinical features, such as hypertrichosis, and further described the neurological features and EEG results. CONCLUSION: This study of a nationwide cohort of individuals with TBRS confirms previously published data and provides additional information and clarifies clinical features to facilitate diagnosis and improve care. This study adds value to the growing body of knowledge on TBRS and broadens its clinical and molecular spectrum.

Variants in FGF10 cause early onset of severe childhood interstitial lung disease: A detailed description of four affected children.

INTRODUCTION: Fibroblast growth factor 10 (FGF10) is a signaling molecule with a well-established role for lung branching morphogenesis. Rare heterozygous, deleterious variants in the FGF10 gene are known causes of the lacrimo-auriculo-dento-digital (LADD) syndrome and aplasia of lacrimal and salivary glands. Previous studies indicate that pathogenic variants in FGF10 can cause childhood Interstitial Lung Disease (chILD) due to severe diffuse developmental disorders of the lung, but detailed reports on clinical presentation and follow-up of affected children are lacking. METHODS: We describe four children with postnatal onset of chILD and heterozygous variants in FGF10, each detected by exome or whole genome sequencing. RESULTS: All children presented with postnatal respiratory failure. Two children died within the first 2 days of life, one patient died at age of 12 years due to right heart failure related to severe pulmonary hypertension (PH) and one patient is alive at age of 6 years, but still symptomatic. Histopathological analysis of lung biopsies from the two children with early postpartum demise revealed diffuse developmental disorder representing acinar dysplasia and interstitial fibrosis. Sequential biopsies of the child with survival until the age of 12 years revealed alveolar simplification and progressive interstitial fibrosis. DISCUSSION: Our report extends the phenotype of FGF10-related disorders to early onset chILD with progressive interstitial lung fibrosis and PH. Therefore, FGF10-related disorder should be considered even without previously described syndromic stigmata in children with postnatal respiratory distress, not only when leading to death in the neonatal period but also in case of persistent respiratory complaints and PH.

TSPEAR variants are primarily associated with ectodermal dysplasia and tooth agenesis but not hearing loss: A novel cohort study.

Biallelic loss-of-function variants in the thrombospondin-type laminin G domain and epilepsy-associated repeats (TSPEAR) gene have recently been associated with ectodermal dysplasia and hearing loss. The first reports describing a TSPEAR disease association identified this gene is a cause of nonsyndromic hearing loss, but subsequent reports involving additional affected families have questioned this evidence and suggested a stronger association with ectodermal dysplasia. To clarify genotype-phenotype associations for TSPEAR variants, we characterized 13 individuals with biallelic TSPEAR variants. Individuals underwent either exome sequencing or panel-based genetic testing. Nearly all of these newly reported individuals (11/13) have phenotypes that include tooth agenesis or ectodermal dysplasia, while three newly reported individuals have hearing loss. Of the individuals displaying hearing loss, all have additional variants in other hearing-loss-associated genes, specifically TMPRSS3, GJB2, and GJB6, that present competing candidates for their hearing loss phenotype. When presented alongside previous reports, the overall evidence supports the association of TSPEAR variants with ectodermal dysplasia and tooth agenesis features but creates significant doubt as to whether TSPEAR variants are a monogenic cause of hearing loss. Further functional evidence is needed to evaluate this phenotypic association.

Skraban-Deardorff syndrome: Six new cases of WDR26-related disease and expansion of the clinical phenotype.

Skraban-Deardorff syndrome (a disease related to variations in the WDR26 gene; OMIM #617616) was first described in a cohort of 15 individuals in 2017. The syndrome comprises intellectual deficiency, severe speech impairment, ataxic gait, seizures, mild hypotonia with feeding difficulties during infancy, and dysmorphic features. Here, we report on six novel heterozygous de novo pathogenic variants in WDR26 in six probands. The patients' phenotypes were consistent with original publication. One patient displayed marked hypotonia with an abnormal muscle biopsy; this finding warrants further investigation. Gait must be closely monitored, in order to highlight any musculoskeletal or neurological abnormalities and prompt further examinations. Speech therapy and alternative communication methods should be initiated early in the clinical follow-up, in order to improve language and oral eating and drinking.

Primrose syndrome: a phenotypic comparison of patients with a ZBTB20 missense variant versus a 3q13.31 microdeletion including ZBTB20.

Primrose syndrome is characterized by variable intellectual deficiency, behavior disorders, facial features with macrocephaly, and a progressive phenotype with hearing loss and ectopic calcifications, distal muscle wasting, and contractures. In 2014, ZBTB20 variants were identified as responsible for this syndrome. Indeed, ZBTB20 plays an important role in cognition, memory, learning processes, and has a transcription repressive effect on numerous genes. A more severe phenotype was discussed in patients with missense single nucleotide variants than in those with large deletions. Here, we report on the clinical and molecular results of 14 patients: 6 carrying ZBTB20 missense SNVs, 1 carrying an early truncating indel, and 7 carrying 3q13.31 deletions, recruited through the AnDDI-Rares network. We compared their phenotypes and reviewed the data of the literature, in order to establish more powerful phenotype-genotype correlations. All 57 patients presented mild-to-severe ID and/or a psychomotor delay. Facial features were similar with macrocephaly, prominent forehead, downslanting palpebral fissures, ptosis, and large ears. Hearing loss was far more frequent in patients with missense SNVs (p = 0.002), ectopic calcification, progressive muscular wasting, and contractures were observed only in patients with missense SNVs (p nonsignificant). Corpus callosum dysgenesis (p = 0.00004), hypothyroidism (p = 0.047), and diabetes were also more frequent in this group. However, the median age was 9.4 years in patients with deletions and truncating variant compared with 15.1 years in those with missense SNVs. Longer follow-up will be necessary to determine whether the phenotype of patients with deletions is also progressive.

De novo TBR1 variants cause a neurocognitive phenotype with ID and autistic traits: report of 25 new individuals and review of the literature.

TBR1, a T-box transcription factor expressed in the cerebral cortex, regulates the expression of several candidate genes for autism spectrum disorders (ASD). Although TBR1 has been reported as a high-confidence risk gene for ASD and intellectual disability (ID) in functional and clinical reports since 2011, TBR1 has only recently been recorded as a human disease gene in the OMIM database. Currently, the neurodevelopmental disorders and structural brain anomalies associated with TBR1 variants are not well characterized. Through international data sharing, we collected data from 25 unreported individuals and compared them with data from the literature. We evaluated structural brain anomalies in seven individuals by analysis of MRI images, and compared these with anomalies observed in TBR1 mutant mice. The phenotype included ID in all individuals, associated to autistic traits in 76% of them. No recognizable facial phenotype could be identified. MRI analysis revealed a reduction of the anterior commissure and suggested new features including dysplastic hippocampus and subtle neocortical dysgenesis. This report supports the role of TBR1 in ID associated with autistic traits and suggests new structural brain malformations in humans. We hope this work will help geneticists to interpret TBR1 variants and diagnose ASD probands.

De novo loss-of-function KCNMA1 variants are associated with a new multiple malformation syndrome and a broad spectrum of developmental and neurological phenotypes.

KCNMA1 encodes the large-conductance Ca2+- and voltage-activated K+ (BK) potassium channel α-subunit, and pathogenic gain-of-function variants in this gene have been associated with a dominant form of generalized epilepsy and paroxysmal dyskinesia. Here, we genetically and functionally characterize eight novel loss-of-function (LoF) variants of KCNMA1. Genome or exome sequencing and the participation in the international Matchmaker Exchange effort allowed for the identification of novel KCNMA1 variants. Patch clamping was used to assess functionality of mutant BK channels. The KCNMA1 variants p.(Ser351Tyr), p.(Gly356Arg), p.(Gly375Arg), p.(Asn449fs) and p.(Ile663Val) abolished the BK current, whereas p.(Cys413Tyr) and p.(Pro805Leu) reduced the BK current amplitude and shifted the activation curves toward positive potentials. The p.(Asp984Asn) variant reduced the current amplitude without affecting kinetics. A phenotypic analysis of the patients carrying the recurrent p.(Gly375Arg) de novo missense LoF variant revealed a novel syndromic neurodevelopmental disorder associated with severe developmental delay, visceral and cardiac malformations, connective tissue presentations with arterial involvement, bone dysplasia and characteristic dysmorphic features. Patients with other LoF variants presented with neurological and developmental symptoms including developmental delay, intellectual disability, ataxia, axial hypotonia, cerebral atrophy and speech delay/apraxia/dysarthria. Therefore, LoF KCNMA1 variants are associated with a new syndrome characterized by a broad spectrum of neurological phenotypes and developmental disorders. LoF variants of KCNMA1 cause a new syndrome distinctly different from gain-of-function variants in the same gene.

Protocol GenoDENT: Implementation of a New NGS Panel for Molecular Diagnosis of Genetic Disorders with Orodental Involvement.

Rare genetic disorders are often challenging to diagnose. Anomalies of tooth number, shape, size, mineralized tissue structure, eruption, and resorption may exist as isolated symptoms or diseases but are often part of the clinical synopsis of numerous syndromes (Bloch-Zupan A, Sedano H, Scully C. Dento/oro/craniofacial anomalies and genetics, 1st edn. Elsevier, Boston, MA, 2012). Concerning amelogenesis imperfecta (AI), for example, mutations in a number of genes have been reported to cause isolated AI, including AMELX, ENAM, KLK4, MMP20, FAM83H, WDR72, C4orf26, SLC24A4, and LAMB3. In addition, many other genes such as DLX3, CNNM4, ROGDI, FAM20A, STIM1, ORAI1, and LTBP3 have been shown to be involved in developmental syndromes with enamel defects. The clinical presentation of the enamel phenotype (hypoplastic, hypomineralized, hypomature, or a combination of severities) alone does not allow a reliable prediction of possible causative genetic mutations. Understanding the potential genetic cause(s) of rare diseases is critical for overall health management of affected patient. One effective strategy to reach a genetic diagnosis is to sequence a selected gene panel chosen for a determined range of phenotypes. Here we describe a laboratory protocol to set up a specific gene panel for orodental diseases.

Homozygous Truncating Variants in TBC1D23 Cause Pontocerebellar Hypoplasia and Alter Cortical Development.

Pontocerebellar hypoplasia (PCH) is a heterogeneous group of rare recessive disorders with prenatal onset, characterized by hypoplasia of pons and cerebellum. Mutations in a small number of genes have been reported to cause PCH, and the vast majority of PCH cases are explained by mutations in TSEN54, which encodes a subunit of the tRNA splicing endonuclease complex. Here we report three families with homozygous truncating mutations in TBC1D23 who display moderate to severe intellectual disability and microcephaly. MRI data from available affected subjects revealed PCH, small normally proportioned cerebellum, and corpus callosum anomalies. Furthermore, through in utero electroporation, we show that downregulation of TBC1D23 affects cortical neuron positioning. TBC1D23 is a member of the Tre2-Bub2-Cdc16 (TBC) domain-containing RAB-specific GTPase-activating proteins (TBC/RABGAPs). Members of this protein family negatively regulate RAB proteins and modulate the signaling between RABs and other small GTPases, some of which have a crucial role in the trafficking of intracellular vesicles and are involved in neurological disorders. Here, we demonstrate that dense core vesicles and lysosomal trafficking dynamics are affected in fibroblasts harboring TBC1D23 mutation. We propose that mutations in TBC1D23 are responsible for a form of PCH with small, normally proportioned cerebellum and should be screened in individuals with syndromic pontocereballar hypoplasia.

Novel de novo mutations in ZBTB20 in Primrose syndrome with congenital hypothyroidism.

The cardinal features of Primrose syndrome (MIM 259050) are dysmorphic facial features, macrocephaly, and intellectual disability, as well as large body size, height and weight, and calcified pinnae. A variety of neurological signs and symptoms have been reported including hearing loss, autism, behavioral abormalities, hypotonia, cerebral calcifications, and hypoplasia of the corpus callosum. Recently, heterozygous de novo missense mutations in ZBTB20, coding for a zing finger protein, have been identified in Primrose syndrome patients. We report a boy with intellectual disability carrying two de novo missense mutations in the last exon of ZBTB20 (Ser616Phe and Gly741Arg; both previously unreported). One of them, Ser616Phe, affects an amino acid located in one of the C2H2 zing-fingers involved in DNA-binding and close to other missense mutations already described. Reverse phenotyping showed that this patient presents with classic features of Primrose syndrome (dysmorphic facies, macrocephaly, hearing loss, hypotonia, hypoplasia of the corpus callosum) and, in addition, congenital hypothyroidism. Review of the literature reveals another Primrose syndrome patient with hypothyroidism and thus, this may represent an under recognized component that should be investigated in other patients. © 2016 Wiley Periodicals, Inc.

Microvillous atrophy: atypical presentations.

OBJECTIVES: Microvillous inclusion disease (MVID) is a cause of intractable diarrhea in infancy. In its classic form, the disease is characterized by a severe persistent watery diarrhea starting within the first days of life. Parenteral nutrition and small bowel transplantation are the only known treatments for the affected children. Histologically, periodic acid-Schiff (PAS) staining shows accumulation of periodic acid-Schiff-positive staining material along the apical pole of enterocytes, whereas transmission electron microscopy exhibits microvillus inclusion bodies within the cytoplasm of enterocytes with rarefied and shortened microvilli and secretory granules. The objective of this work was to explore clinical, morphological, and genetic findings in cases of MVID with unusual presentations. METHODS: Clinical, histological, and genetic findings are reported for 8 cases of MVID with atypical presentation. RESULTS: The diarrhea started after several months in 3 cases. It was usually less abundant and 3 patients were weaned off parenteral nutrition. None required intestinal transplantation. Three patients experienced malformations, dysmorphy, sensory disabilities, and severe mental retardation. One had a hydrocephaly. Three patients had a cholestasis with low γ-glutamyl transferase levels. Light microscopy showed histological abnormalities consistent with MVID in all of the cases, but the lesions were sometimes focal or delayed. Transmission electron microscopy retrieved some criteria of MVID in 4 patients. Finally, 6 patients were homozygotes or compound heterozygotes for MYO5B mutations. CONCLUSIONS: This study extends the spectrum of MVID to less severe clinical presentations.

CFTR p.Arg117His associated with CBAVD and other CFTR-related disorders.

BACKGROUND: The high frequency of the cystic fibrosis (CF) transmembrane conductance regulator (CFTR) gene mutation p.Arg117His in patients with congenital bilateral absence of the vas deferens (CBAVD) and in newborns screened for CF has created a dilemma. METHODS: Phenotypic and genotypic data were retrospectively collected in 179 non-newborn French individuals carrying p.Arg117His and a second CFTR mutation referred for symptoms or family history, by all French molecular genetics laboratories, referring physicians, CF care centres and infertility clinics. RESULTS: 97% of the patients had the intronic T7 normal variant in cis with p.Arg117His. 89% patients were male, with CBAVD being the reason for referral in 76%. In 166/179 patients with available detailed clinical features, final diagnoses were: four late-onset marked pulmonary disease, 83 isolated CBAVD, 67 other CFTR-related phenotypes, including 44 CBAVD with pulmonary and/or pancreatic symptoms and 12 asymptomatic cases. Respiratory symptoms were observed in 30% of the patients, but the overall phenotype was mild. No correlation was observed between sweat chloride concentrations and disease severity. Five couples at risk of CF offspring were identified and four benefited from prenatal or preimplantation genetic diagnoses (PND or PGD). Eight children were born, including four who were compound heterozygous for p.Arg117His and one with a severe CF mutation. CONCLUSIONS: Patients with CBAVD carrying p.Arg117His and a severe CF mutation should benefit from a clinical evaluation and follow-up. Depending on the CBAVD patients' genotype, a CFTR analysis should be considered in their partners in order to identify CF carrier couples and offer PND or PGD.

Sirenomelia and caudal malformations in two families.

We report on two families with co-occurrence of sirenomelia and caudal malformations. In the first family, the mother had undergone surgery for a short form of imperforate anus. Her first pregnancy was terminated because of bilateral renal agenesis with oligohydramnios. Her second pregnancy was interrupted because of sirenomelia. The second family was referred to us because of caudal malformation in their two children. The parents' spinal radiographs were normal. The first pregnancy resulted in a girl with imperforate anus, absence of S3-S5 and coccyx, abnormal pelvic floor, and an almost bifid anteriorly located bladder. The second pregnancy resulted in a baby girl with sirenomelia. No diabetes was present during the pregnancies in either of these two families. These families confirm the hypothesis that major genes are responsible for the embryogenesis of the caudal part of the embryo, with variable expression, as has been already described in sirenomelia mouse models (CYP26A1, BMP7/tsg). Molecular studies are underway in these families and in sporadic cases in our laboratory to explore the genetic basis of sirenomelia in humans.

Many roads lead to primary autosomal recessive microcephaly.

Autosomal recessive primary microcephaly (MCPH), historically referred to as Microcephalia vera, is a genetically and clinically heterogeneous disease. Patients with MCPH typically exhibit congenital microcephaly as well as mental retardation, but usually no further neurological findings or malformations. Their microcephaly with grossly preserved macroscopic organization of the brain is a consequence of a reduced brain volume, which is evident particularly within the cerebral cortex and thus results to a large part from a reduction of grey matter. Some patients with MCPH further provide evidence of neuronal heterotopias, polymicrogyria or cortical dysplasia suggesting an associated neuronal migration defect. Genetic causes of MCPH subtypes 1-7 include mutations in genes encoding microcephalin, cyclin-dependent kinase 5 regulatory associated protein 2 (CDK5RAP2), abnormal spindle-like, microcephaly associated protein (ASPM), centromeric protein J (CENPJ), and SCL/TAL1-interrupting locus (STIL) as well as linkage to the two loci 19q13.1-13.2 and 15q15-q21. Here, we provide a timely overview of current knowledge on mechanisms leading to microcephaly in humans with MCPH and abnormalities in cell division/cell survival in corresponding animal models. Understanding the pathomechanisms leading to MCPH is of high importance not only for our understanding of physiologic brain development (particularly of cortex formation), but also for that of trends in mammalian evolution with a massive increase in size of the cerebral cortex in primates, of microcephalies of other etiologies including environmentally induced microcephalies, and of cancer formation.

Amniotic fluid digestive enzyme analysis is useful for identifying CFTR gene mutations of unclear significance.

BACKGROUND: The large number of CFTR [cystic fibrosis transmembrane conductance regulator (ATP-binding cassette sub-family C, member 7)] mutations and the existence of variants of unclear significance complicate the prenatal diagnosis of cystic fibrosis (CF). The aim of this study was to determine whether the pattern of amniotic fluid digestive enzymes (AF-DEs) could be correlated with the severity of CFTR mutations. METHODS: The AF-DE pattern (gamma-glutamyltranspeptidase, aminopeptidase M, and the intestinal isoform of alkaline phosphatase) was retrospectively analyzed in 43 AF samples. All fetuses presented 2 CFTR mutations, which were classified according to the severity of the disease: CF/CF (n = 38); CF/CFTR-related disorders (n = 1); and CF/unknown variant (n = 4). The relationships between clinical CF status, CFTR mutations, and AF-DE pattern were studied. RESULTS: Of 38 severely affected CF fetuses, an "obstructive" AF-DE pattern was observed in 15 of 15 samples collected before 22 weeks, irrespective of the CFTR mutation (diagnostic sensitivity, 100%; diagnostic specificity, 99.8%). In the 23 fetuses evaluated after 22 weeks, the AF-DE pattern was abnormal in 7 cases and noncontributive in 16 (diagnostic sensitivity, 30.4%; diagnostic specificity, 99.8%). Of the 5 questionable cases (F508del/N1224K, F508del/L73F, 3849+10kbC>T/G1127E, F508del/S1235R, F508del/G622D), all were CF symptom free at 2-4 years of follow-up. The AF-DE pattern (<22 weeks) was typical in 3 cases but abnormal in the last 2 cases. CONCLUSIONS: AF-DE analysis is of value for prenatal CF diagnosis in classic forms of CF and could be helpful in nonclassic CF.

NHERF1 mutations and responsiveness of renal parathyroid hormone.

Impaired renal phosphate reabsorption, as measured by dividing the tubular maximal reabsorption of phosphate by the glomerular filtration rate (TmP/GFR), increases the risks of nephrolithiasis and bone demineralization. Data from animal models suggest that sodium-hydrogen exchanger regulatory factor 1 (NHERF1) controls renal phosphate transport. We sequenced the NHERF1 gene in 158 patients, 94 of whom had either nephrolithiasis or bone demineralization. We identified three distinct mutations in seven patients with a low TmP/GFR value. No patients with normal TmP/GFR values had mutations. The mutants expressed in cultured renal cells increased the generation of cyclic AMP (cAMP) by parathyroid hormone (PTH) and inhibited phosphate transport. These NHERF1 mutations suggest a previously unrecognized cause of renal phosphate loss in humans.

Molecular epidemiology of chronic granulomatous disease in a series of 80 kindreds: identification of 31 novel mutations.

Chronic granulomatous disease (CGD) results from constitutional inactivating mutations in the CYBB, NCF1, CYBA or NCF2 genes that encode subunits of phagocyte NADPH oxidase. We report the findings of molecular analysis of 80 kindred. In 75 unrelated male and 5 female probands, CGD was suspected on the basis of clinical symptoms, and biological samples were referred to our laboratory between 2000 and 2007. Seventy seven patients were found to have mutations in CYBB, NCF1, CYBA or NCF2 (52 different mutations including 31 mutations not previously reported). CYBB was the most frequently mutated gene (58 males and 3 females, 76%). In autosomal recessive forms of the disease, mutations were found in NCF1 (11 patients), NCF2 (3 patients) and CYBA (2 patients). We observed that significantly fewer females were affected by autosomal recessive CGD than expected (2 females/14 males; p=0.002), suggesting that female patients with CGD may be under diagnosed.

PTHR1 mutations associated with Ollier disease result in receptor loss of function.

PTHR1-signaling pathway is critical for the regulation of endochondral ossification. Thus, abnormalities in genes belonging to this pathway could potentially participate in the pathogenesis of Ollier disease/Maffucci syndrome, two developmental disorders defined by the presence of multiple enchondromas. In agreement, a functionally deleterious mutation in PTHR1 (p.R150C) was identified in enchondromas from two of six unrelated patients with enchondromatosis. However, neither the p.R150C mutation (26 tumors) nor any other mutation in the PTHR1 gene (11 patients) could be identified in another study. To further define the role of PTHR1-signaling pathway in Ollier disease and Maffucci syndrome, we analyzed the coding sequences of four genes (PTHR1, IHH, PTHrP and GNAS1) in leucocyte and/or tumor DNA from 61 and 23 patients affected with Ollier disease or Maffucci syndrome, respectively. We identified three previously undescribed missense mutations in PTHR1 in patients with Ollier disease at the heterozygous state. Two mutations (p.G121E, p.A122T) were present only in enchondromas, and one (p.R255H) in both enchondroma and leukocyte DNA. Assessment of receptor function demonstrated that these three mutations impair PTHR1 function by reducing either the affinity of the receptor for PTH or the receptor expression at the cell surface. These mutations were not found in DNA from 222 controls. Including our data, PTHR1 functionally deleterious mutations have now been identified in five out 31 enchondromas from Ollier patients. These findings provide further support for the idea that heterozygous mutations in PTHR1 that impair receptor function participate in the pathogenesis of Ollier disease in some patients.

Severe X-linked chronic granulomatous disease in two unrelated females.

Chronic granulomatous disease (CGD) is a rare primary immunodeficiency caused by mutations of one of the subunits of phagocyte reduced nicotinamide adenine dinucleotide phosphate (NADPH) oxidase leading to decreased or complete absence of neutrophil oxidative burst. We report the clinical and laboratory findings in two young unrelated females 14 and 9 years of age and natives of Tahiti and Reunion Islands, respectively, with severe X-linked granulomatous disease. In both cases, the infectious pattern was unusual, with convergent symptoms suggesting underlying mycobacterial infection. Functional analysis revealed low residual NADPH oxidase activity with about 5-10% of normal neutrophil population. De novo null mutations affecting the CYBB gene that encodes the gp91 protein were found in both cases in the heterozygous state (in patient 1, p.Arg130X in exon 5, and in patient 2, a novel insertion in exon 6, c.632_633insCATC). Methylation analysis confirmed that phenotype expression was linked to skewed X inactivation and showed that the de novo mutation arose on the maternally inherited chromosome in one case and on the paternally inherited chromosome in the other case. In conclusion, X-linked CGD carriers could therefore be at risk for severe infectious diseases depending on the skewed X inactivation pattern and the infectious context.