Microcephaly, intractable seizures and developmental delay caused by biallelic variants in TBCD: further delineation of a new chaperone-mediated tubulinopathy.

Microtubule dynamics play a crucial role in neuronal development and function, and several neurodevelopmental disorders have been linked to mutations in genes encoding tubulins and functionally related proteins. Most recently, variants in the tubulin cofactor D (TBCD) gene, which encodes one of the five co-chaperones required for assembly and disassembly of α/ß-tubulin heterodimer, were reported to underlie a recessive neurodevelopmental/neurodegenerative disorder. We report on five patients from three unrelated families, who presented with microcephaly, intellectual disability, intractable seizures, optic nerve pallor/atrophy, and cortical atrophy with delayed myelination and thinned corpus callosum on brain imaging. Exome sequencing allowed the identification of biallelic variants in TBCD segregating with the disease in the three families. TBCD protein level was significantly reduced in cultured fibroblasts from one patient, supporting defective TBCD function as the event underlying the disorder. Such reduced expression was associated with accelerated microtubule re-polymerization. Morpholino-mediated TBCD knockdown in zebrafish recapitulated several key pathological features of the human disease, and TBCD overexpression in the same model confirmed previous studies documenting an obligate dependency on proper TBCD levels during development. Our findings confirm the link between inactivating TBCD variants and this newly described chaperone-associated tubulinopathy, and provide insights into the phenotype of this disorder.

Identification of the gene causing mucolipidosis type IV.

Mucolipidosis type IV (MLIV) is an autosomal recessive, neurodegenerative, lysosomal storage disorder characterized by psychomotor retardation and ophthalmological abnormalities including corneal opacities, retinal degeneration and strabismus. Most patients reach a maximal developmental level of 12?15 months. The disease was classified as a mucolipidosis following observations by electron microscopy indicating the lysosomal storage of lipids together with water-soluble, granulated substances. Over 80% of the MLIV patients diagnosed are Ashkenazi Jews, including severely affected and mildly affected patients. The gene causing MLIV was previously mapped to human chromosome 19p13.2-13.3 in a region of approximately 1 cM (ref. 7). Haplotype analysis in the MLIV gene region of over 70 MLIV Ashkenazi chromosomes indicated the existence of two founder chromosomes among 95% of the Ashkenazi MLIV families: a major haplotype in 72% and a minor haplotype in 23% of the MLIV chromosomes (ref. 7, and G.B., unpublished data). The remaining 5% are distinct haplotypes found only in single patients. The basic metabolic defect causing the lysosomal storage in MLIV has not yet been identified. Thus, positional cloning was an alternative to identify the MLIV gene. We report here the identification of a new gene in this human chromosomal region in which MLIV-specific mutations were identified.

Identification of the gene that, when mutated, causes the human obesity syndrome BBS4.

Bardet-Biedl syndrome (BBS, MIM 209900) is a heterogeneous autosomal recessive disorder characterized by obesity, pigmentary retinopathy, polydactyly, renal malformations, mental retardation, and hypogenitalism. The disorder is also associated with diabetes mellitus, hypertension, and congenital heart disease. Six distinct BBS loci map to 11q13 (BBS1), 16q21 (BBS2), 3p13-p12 (BBS3), 15q22.3-q23 (BBS4), 2q31 (BBS5), and 20p12 (BBS6). Although BBS is rare in the general population (<1/100,000), there is considerable interest in identifying the genes causing BBS because components of the phenotype, such as obesity and diabetes, are common. We and others have demonstrated that BBS6 is caused by mutations in the gene MKKS (refs. 12,13), mutation of which also causes McKusick-Kaufman syndrome (hydrometrocolpos, post-axial polydactyly, and congenital heart defects). MKKS has sequence homology to the alpha subunit of a prokaryotic chaperonin in the thermosome Thermoplasma acidophilum. We recently identified a novel gene that causes BBS2. The BBS2 protein has no significant similarity to other chaperonins or known proteins. Here we report the positional cloning and identification of mutations in BBS patients in a novel gene designated BBS4.

Origin and spread of a common deletion causing mucolipidosis type II: insights from patterns of haplotypic diversity.

Mucolipidosis II (ML II alpha/beta), or I-cell disease, is a rare genetic disease in which activity of the uridine diphosphate (UDP)-N-acetylglucosamine:lysosomal enzyme N-acetylglucosamine-1-phosphotransferase (GlcNAc-phosphotransferase) is absent. GlcNAc-phosphotransferase is a multimeric enzyme encoded by two genes, GNPTAB and GNPTG. A spectrum of mutations in GNPTAB has been recently reported to cause ML II alpha/beta. Most of these mutations were found to be private or rare. However, the mutation c.3503_3504delTC has been detected among Israeli and Palestinian Arab-Muslim, Turkish, Canadian, Italian, Portuguese, Irish traveller and US patients. We analysed 44 patients who were either homozygous or compound heterozygous for this deletion (22 Italians, 8 Arab-Muslims, 1 Turk, 3 Argentineans, 3 Brazilians, 2 Irish travellers and 5 Portuguese) and 16 carriers (15 Canadians and 1 Italian) for three intragenic polymorphisms: c.-41_-39delGGC, c.18G>A and c.1932A>G as well as two microsatellite markers flanking the GNPTAB gene (D12S1607 and D12S1727). We identified a common haplotype in all chromosomes bearing the c.3503_3504delTC mutation. In summary, we showed that patients carrying the c.3503_3504delTC deletion presented with a common haplotype, which implies a common origin of this mutation. Additionally, the level of diversity observed at the most distant locus indicates that the mutation is relatively ancient (around 2063 years old), and the geographical distribution further suggests that it probably arose in a peri-Mediterranean region.

Phenotypic variability in 49 cases of ESCO2 mutations, including novel missense and codon deletion in the acetyltransferase domain, correlates with ESCO2 expression and establishes the clinical criteria for Roberts syndrome.

BACKGROUND: Roberts syndrome (RBS) and SC phocomelia are caused by mutations in ESCO2, which codes for an acetyltransferase involved in the regulation of sister chromatid cohesion. Of 26 mutations described to date, only one missense mutation has been reported and all others are predicted to be truncating mutations. Genotype-phenotype analysis has been hampered by limited numbers of patients with clinical information available. OBJECTIVE: To provide unpublished clinical data for 31 patients with proven ESCO2 mutations and combine this series with previously reported clinical and mutation data on 18 cases. Methods Genotype-phenotype correlations and functional effects of two novel ESCO2 mutations were analysed. In situ hybridisation on human embryos at Carnegie stages 14, 17 and 21 was performed to study ESCO2 expression during development. RESULTS AND CONCLUSIONS: Using the cohort of 49 patients, the clinical criteria for RBS were delineated to include: growth retardation; symmetric mesomelic shortening of the limbs in which the upper limbs are more commonly and severely affected than the lower limbs; characteristic facies with microcephaly. The severity of malformations of the facies correlates with the severity of limb reduction. The occurrence of corneal opacities may be associated with specific mutations. Two new mutations, both in the ESCO2 acetyltransferase domain, are described and their acetylation effects in vitro demonstrated. In situ hybridisation on human embryos showed ESCO2 expression in the brain, face, limb, kidney and gonads, which corresponds to the structures affected in RBS.

Mutations in the mitochondrial glutamate carrier SLC25A22 in neonatal epileptic encephalopathy with suppression bursts.

Neonatal epileptic encephalopathies with suppression bursts (SBs) are very severe and relatively rare diseases characterized by neonatal onset of seizures, interictal electroencephalogram (EEG) with SB pattern and very poor neurological outcome or death. Their etiology remains elusive but they are occasionally caused by metabolic diseases or malformations. Studying an Arab Muslim Israeli consanguineous family, with four affected children presenting a severe neonatal epileptic encephalopathy, we have previously identified a mutation in the SLC25A22 gene encoding a mitochondrial glutamate transporter. In this report, we describe a novel SLC25A22 mutation in an unrelated patient born from first cousin Algerian parents and presenting severe epileptic encephalopathy characterized by an EEG with SB, hypotonia, microcephaly and abnormal electroretinogram. We showed that this patient carried a homozygous p.G236W SLC25A22 mutation which alters a highly conserved amino acid and completely abolishes the glutamate carrier's activity in vitro. Comparison of the clinical features of patients from both families suggests that SLC25A22 mutations are responsible for a novel clinically recognizable epileptic encephalopathy with SB.

Novel WWOX deleterious variants cause early infantile epileptic encephalopathy, severe developmental delay and dysmorphism among Yemenite Jews.

The human WW Domain Containing Oxidoreductase (WWOX) gene was originally described as a tumor suppressor gene. However, recent reports have demonstrated its cardinal role in the pathogenesis of central nervous systems disorders such as epileptic encephalopathy, intellectual disability, and spinocerebellar ataxia. We report on six patients from three unrelated families of full or partial Yemenite Jewish ancestry exhibiting early infantile epileptic encephalopathy and profound developmental delay. Importantly, four patients demonstrated facial dysmorphism. Exome sequencing revealed that four of the patients were homozygous for a novel WWOX c.517-2A > G splice-site variant and two were compound heterozygous for this variant and a novel c.689A > C, p.Gln230Pro missense variant. Complementary DNA sequencing demonstrated that the WWOX c.517-2A > G splice-site variant causes skipping of exon six. A carrier rate of 1:177 was found among Yemenite Jews. We provide the first detailed description of patients harboring a splice-site variant in the WWOX gene and propose that the clinical synopsis of WWOX related epileptic encephalopathy should be broadened to include facial dysmorphism. The increased frequency of the c.517-2A > G splice-site variant among Yemenite Jews coupled with the severity of the phenotype makes it a candidate for inclusion in expanded preconception screening programs.

Home-based infusion therapy for patients with Fabry disease.

Fabry disease is an inherited, progressive, life-threatening disease; therefore, lifelong therapy is needed. By replacing the deficient enzyme, disease progression may be delayed or halted, thereby avoiding serious complications. Hospital-based agalsidase therapy is generally perceived as inconvenient and home-based infusion therapy is greatly appreciated by patients, their families and healthcare professionals. Patients can get familiar with infusion therapy in a hospital setting and, if specific requirements are fulfilled, routine nurse-assisted infusion, or self-care, at the patient's home can be organized. A stable patient who tolerates the infusion and a suitable home environment are prerequisites for home therapy. The authors' clinical experiences underscore the safety and practicality of home therapy. In addition to a major positive impact on the patient's quality of life, home infusion therapy may reduce the constraints of hospital resources. This article reviews the collective experiences with agalsidase beta home infusion therapy and outlines how safe, patient-centred homecare can be organized. Home infusion therapy with Fabrazyme should not be withheld from patients considered eligible according to the proposed criteria. Similar approaches to other enzyme therapies are also possible.

Molecular basis of variant pseudo-hurler polydystrophy (mucolipidosis IIIC)

Mucolipidosis IIIC, or variant pseudo-Hurler polydystrophy, is an autosomal recessive disease of lysosomal hydrolase trafficking. Unlike the related diseases, mucolipidosis II and IIIA, the enzyme affected in mucolipidosis IIIC (N-Acetylglucosamine-1-phosphotransferase [GlcNAc-phosphotransferase]) retains full transferase activity on synthetic substrates but lacks activity on lysosomal hydrolases. Bovine GlcNAc-phosphotransferase has recently been isolated as a multisubunit enzyme with the subunit structure alpha(2)beta(2)gamma(2). We cloned the cDNA for the human gamma-subunit and localized its gene to chromosome 16p. We also showed, in a large multiplex Druze family that exhibits this disorder, that MLIIIC also maps to this chromosomal region. Sequence analysis of the gamma-subunit cDNA in patients from 3 families identified a frameshift mutation, in codon 167 of the gamma subunit, that segregated with the disease, indicating MLIIIC results from mutations in the phosphotransferase gamma-subunit gene. This is to our knowledge the first description of the molecular basis for a human mucolipidosis and suggests that the gamma subunit functions in lysosomal hydrolase recognition.

Mucolipidosis type IV: novel MCOLN1 mutations in Jewish and non-Jewish patients and the frequency of the disease in the Ashkenazi Jewish population.

The gene MCOLN1 is mutated in Mucolipidosis type IV (MLIV), a neurodegenerative, recessive, lysosomal storage disorder. The disease is found in relatively high frequency among Ashkenazi Jews due to two founder mutations that comprise 95% of the MLIV alleles in this population [Bargal et al., 2000]. In this report we complete the mutation analysis of Jewish and non-Jewish MLIV patients whose DNA were available to us. Four novel mutations were identified in the MCOLN1 gene of severely affected patients: two missense, T232P and F465L; a nonsense, R322X; and an 11-bp insertion in exon 12. The nonsense mutation (R322X) was identified in two unrelated patients with different haplotypes in the MCOLN1 chromosomal region, indicating a mutation hotspot in this CpG site. An in-frame deletion (F408del) was identified in a patient with unusual mild psychomotor retardation. The frequency of MLIV in the general Jewish Ashkenazi population was estimated in a sample of 2,000 anonymous, unrelated individuals assayed for the two founder mutations. This analysis indicated a heterozygotes frequency of about 1/100. A preferred nucleotide numbering system for MCOLN1 mutations is presented and the issue of a screening program for the detection of high-risk families in the Jewish Ashkenazi population is discussed.

Mucolipidosis type IV: the origin of the disease in the Ashkenazi Jewish population.

Mucolipidosis type IV (MLIV) is a neurodegenerative lysosomal storage disease in which most of the patients diagnosed hitherto are Ashkenazi Jews. The basic metabolic defect causing this disease is still unknown and the relevant gene has not yet been mapped or cloned. Seventeen Israel Ashkenazi families with MLIV patients had been interviewed to study their family origin. Although the families immigrated to Israel from various European countries they all could trace their roots three to four generations back to northern Poland or the immediate neighbouring country, Lithuania. Furthermore, there are only one or two ultraorthodox families among the 70-80 Ashkenazi families with MLIV patients worldwide, a marked under-representation of this group which constitutes at least 10% of the Ashkenazi population. This data indicate that MLIV mutation occurred only around the 18th and 19th centuries, after the major expansion of this population, in a founder in this defined European region belonging to a more modern, secular family.

Giant congenital aortic aneurysm with cleft sternum, supraumbilical raphé, and hemangiomatosis: report and review.

We report on a child with giant congenital aortic aneurysm, sternal defect, hemangiomas of face, supraumbilical raphé, and review the only two other cases reported to date. Congenital aortic aneurysm is an ominous malformation that has to be systematically searched in children with the sternal malformation/vascular dysplasia complex.

Contribution of connexin 26 mutations to nonsyndromic deafness in Ashkenazi patients and the variable phenotypic effect of the mutation 167delT.

Twenty-seven unrelated Jewish Ashkenazi patients with nonsyndromic prelingual deafness (NSD) were analyzed for mutations in the coding sequence of the connexin 26 (Cx26) gene. Biallelic mutations were identified in 19 of the 27 patients (70.4%); 12 were homozygous for the mutation 167delT, 2 were homozygous for the mutation 35delG, and 5 were compound 167delT/35delG heterozygotes. In addition three patients were heterozygous with no second identified mutation in the Cx26 gene. Biallelic mutations in the Cx26 gene account for 83% of familial cases and 44% of the sporadic cases. Among 268 unselected Ashkenazi individuals, 20 were 167delT/N heterozygotes, giving an estimate of 7.5% carrier frequency. Based on the 167delT carrier frequency in three studies (including the present one), it is expected that 167delT/167delT homozygotes account for 70% of all patients with NSD (1 in 1300). The hearing capacity of 30 patients (probands and their sibs) with biallelic Cx26 mutations and at least one allele with 167delT demonstrated inter- and intrafamilial variability from profound to mild hearing impairment.

Agenesis of tibia with ectrodactyly/Gollop-Wolfgang complex associated with congenital heart malformations and additional skeletal abnormalities.

We report on a child with bifid femur, absent tibiae, hypoplastic hallux, bilateral club feet, congenital heart defects, and segmentation anomalies of the spine and ribs. Parents are consanguineous, from a region where other consanguineous families with similarly affected individuals have been reported. Clinical and genetic controversies of the tibial aplasia-ectrodactyly syndrome/Gollop-Wolfgang complex are discussed.

Oral ciprofloxacin in the management of chronic suppurative otitis media without cholesteatoma in children: preliminary experience in 21 children.

The current medical management of children with chronic suppurative otitis media without cholesteatoma unresponsive to local treatment and oral antibiotics is intravenous antibiotic therapy in the hospital setting. We studied the efficacy and toxicity of oral ciprofloxacin in chronic suppurative otitis media. Twenty-one children received oral ciprofloxacin, 30 mg/kg/day. Ear discharge was positive for bacteria resistant to other oral medications and susceptible to the quinolones. The mean duration of treatment was 16.7 days. In 18 children suppuration ceased and 3 failed their first course. During a mean follow-up of 15.4 months, 6 children remained free of ear, nose and throat problems. Otorrhea recurred in 12 children. Ear cultures were positive for organisms susceptible to amoxicillin in 5 of them. In 7 cases Pseudomonas aeruginosa was again isolated from otorrhea. Repeated antibiotic therapy was advocated only in 3 (2 responded to ciprofloxacin; 1 failed ciprofloxacin and was cured by ceftazidime). Adverse clinical effects were not observed. Transient neutropenia was observed in 1 child. There was no change in the height percentile. The results of this study show that children with chronic suppurative otitis media without cholesteatoma can be effectively treated with oral ciprofloxacin. This novel approach may prevent hospitalization.

TAT gene mutation analysis in three Palestinian kindreds with oculocutaneous tyrosinaemia type II; characterization of a silent exonic transversion that causes complete missplicing by exon 11 skipping.

Deficiency of the hepatic cytosolic enzyme tyrosine aminotransferase (TAT) causes marked hypertyrosinaemia leading to painful palmoplantar hyperkeratoses, pseudodendritic keratitis and variable mental retardation (oculocutaneous tyrosinaemia type II or Richner-Hanhart syndrome). Parents may therefore seek prenatal diagnosis, but this is not possible by biochemical assays as tyrosine does not accumulate in amniotic fluid and TAT is not expressed in chorionic villi or amniocytes. Molecular analysis is therefore the only possible approach for prenatal diagnosis and carrier detection. To this end, we sought TAT gene mutations in 9 tyrosinaemia II patients from three consanguineous Palestinian kindreds. In two kindreds (7 patients), the only potential abnormality identified after sequencing all 12 exons and exon-intron boundaries was homozygosity for a silent, single-nucleotide transversion c.1224G > T (p.T408T) at the last base of exon 11. This was predicted to disrupt the 5' donor splice site of exon 11 and result in missplicing. However, as TAT is expressed exclusively in liver, patient mRNA could not be obtained for splicing analysis. A minigene approach was therefore used to assess the effect of c.1224G > T on exon 11 splicing. Transfection experiments with wild-type and c.1224G > T mutant minigene constructs demonstrated that c.1224G > T results in complete exon 11 skipping, illustrating the utility of this approach for confirming a putative splicing defect when cDNA is unavailable. Homozygosity for a c.1249C > T (R417X) exon 12 nonsense mutation (previously reported in a French patient) was identified in both patients from the third kindred, enabling successful prenatal diagnosis of an unaffected fetus using chorionic villous tissue.

Hereditary lymphedema type I associated with VEGFR3 mutation: the first de novo case and atypical presentations.

Mutations in the vascular endothelial growth factor receptor 3 gene, VEGFR3/FLT4, have been identified in a subset of families with hereditary lymphedema type I or Milroy disease (MIM 153100). Individuals carrying a VEGFR3 mutation exhibit congenital edema of the lower limbs, usually bilaterally and below the knees, sometimes associated with cellulitis, prominent veins, papillomatosis, upturned toenails, and hydrocele. In this study, we report the first de novo VEGFR3 mutation in a patient with sporadic congenital lymphedema. We also describe three other families with a VEGFR3 mutation. In each family, one individual had an atypical clinical presentation of hereditary lymphedema type I, whereas the others had the classical VEGFR3 mutation-caused phenotype. The atypical presentations included pre-natal pleural effusion, spontaneous resorption of lymphedema and elephantiasis. Three of the four identified mutations were novel. These data show that de novo VEGFR3 mutations may be present in patients without family history of congenital lymphedema. This has implications for follow-up care, as such individuals have nearly a 50% risk for occurrence of lymphedema in their children. Our findings also indicate that although most patients with a VEGFR3 mutation have the well-defined phenotype for hereditary lymphedema type I, there are exceptions that should be considered in genetic counseling. Because VEGFR3 mutation can cause generalized lymphatic dysfunction and can thus result in hydrops fetalis, VEGFR3 screening should be added to the investigation of cases of hydrops fetalis of an unknown etiology.

Negative expansion of the myotonic dystrophy unstable sequence.

We have analyzed the unstable fragment of the myotonic dystrophy (DM) gene in a pregnancy at 50% risk for DM. The affected father in this family had a 3.0-kb expansion of the DM unstable region. The fetus inherited the mutated gene, but with an expansion of 0.5 kb. This case represented a counseling problem in light of the absence of data concerning "negative expansion." Analysis of the DM gene in 17 families with 72 affected individuals revealed four more cases of negative expansions, all of them in paternal transmissions. The possible significance of this finding is discussed.