A case report of two male siblings with autism and duplication of Xq13-q21, a region including three genes predisposing for autism.

Autism spectrum disorder, severe behaviour problems and duplication of the Xq12 to Xq13 region have recently been described in three male relatives. To describe the psychiatric comorbidity and dysmorphic features, including craniosynostosis, of two male siblings with autism and duplication of the Xq13 to Xq21 region, and attempt to narrow down the number of duplicated genes proposed to be leading to global developmental delay and autism. We performed DNA sequencing of certain exons of the TWIST1 gene, the FGFR2 gene and the FGFR3 gene. We also performed microarray analysis of the DNA. In addition to autism, the two male siblings exhibited severe learning disability, self-injurious behaviour, temper tantrums and hyperactivity, and had no communicative language. Chromosomal analyses were normal. Neither of the two siblings showed mutations of the sequenced exons known to produce craniosynostosis. The microarray analysis detected an extra copy of a region on the long arm of chromosome X, chromosome band Xq13.1-q21.1. Comparison of our two cases with previously described patients allowed us to identify three genes predisposing for autism in the duplicated chromosomal region. Sagittal craniosynostosis is also a new finding linked to the duplication.

Novel missense mutations in the glycine receptor ß subunit gene (GLRB) in startle disease.

Startle disease is a rare, potentially fatal neuromotor disorder characterized by exaggerated startle reflexes and hypertonia in response to sudden unexpected auditory, visual or tactile stimuli. Mutations in the GlyR α(1) subunit gene (GLRA1) are the major cause of this disorder, since remarkably few individuals with mutations in the GlyR ß subunit gene (GLRB) have been found to date. Systematic DNA sequencing of GLRB in individuals with hyperekplexia revealed new missense mutations in GLRB, resulting in M177R, L285R and W310C substitutions. The recessive mutation M177R results in the insertion of a positively-charged residue into a hydrophobic pocket in the extracellular domain, resulting in an increased EC(50) and decreased maximal responses of α(1)ß GlyRs. The de novo mutation L285R results in the insertion of a positively-charged side chain into the pore-lining 9' position. Mutations at this site are known to destabilize the channel closed state and produce spontaneously active channels. Consistent with this, we identified a leak conductance associated with spontaneous GlyR activity in cells expressing α(1)ß(L285R) GlyRs. Peak currents were also reduced for α(1)ß(L285R) GlyRs although glycine sensitivity was normal. W310C was predicted to interfere with hydrophobic side-chain stacking between M1, M2 and M3. We found that W310C had no effect on glycine sensitivity, but reduced maximal currents in α(1)ß GlyRs in both homozygous (α(1)ß(W310C)) and heterozygous (α(1)ßß(W310C)) stoichiometries. Since mild startle symptoms were reported in W310C carriers, this may represent an example of incomplete dominance in startle disease, providing a potential genetic explanation for the 'minor' form of hyperekplexia.

Mutations in SPINT2 cause a syndromic form of congenital sodium diarrhea.

Autosomal-recessive congenital sodium diarrhea (CSD) is characterized by perinatal onset of a persistent watery diarrhea with nonproportionally high fecal sodium excretion. Defective jejunal brush-border Na(+)/H(+) exchange has been reported in three sporadic patients, but the molecular basis of the disease has not been elucidated. We reviewed data from a large cohort of CSD patients (n = 24) and distinguished CSD associated with choanal or anal atresia, hypertelorism, and corneal erosions--i.e., a syndromic form of CSD--occurring in ten families from an isolated form--i.e., classic CSD--presenting in seven families. Patients from both groups have a high risk of mortality due to immediate electrolyte imbalances and complications from long-term parenteral nutrition in the first years of life, but survivors can eventually adapt to partial or complete enteral nutrition. A genome-wide SNP scan was applied and identified a homozygous c.593-1G-->A splicing mutation in SPINT2, encoding a Kunitz-type serine-protease inhibitor, in one extended kindred with syndromic CSD. The same mutation and four distinct, homozygous or compound heterozygous mutations (p.Y163C, c.1A-->T, c.337+2T-->C, c.553+2T-->A) were identified in all syndromic patients. No SPINT2 mutations were found in classic-CSD patients. SPINT2 mutations were associated with loss of protein synthesis or failure to inhibit the serine protease trypsin in vitro. We delineate syndromic CSD as a distinct disease entity caused by SPINT2 loss-of-function mutations. SPINT2 mutations might lead to an excess of yet unknown serine protease activity in affected tissues.

Incompletely penetrant PKD1 alleles mimic the renal manifestations of ARPKD.

Autosomal dominant polycystic kidney disease (ADPKD), caused by mutation in PKD1 or PKD2, is usually an adult-onset disorder but can rarely manifest as a neonatal disease within a family characterized by otherwise typical ADPKD. Coinheritance of a hypomorphic PKD1 allele in trans with an inactivating PKD1 allele is one mechanism that can cause early onset ADPKD. Here, we describe two pedigrees without a history of cystic kidney disease that each contain two patients with onset of massive PKD in utero. The presentations were typical of autosomal recessive PKD (ARPKD) but they were not linked to the known ARPKD gene, PKHD1. Mutation analysis of the ADPKD genes provided strong evidence that both families inherited, in trans, two incompletely penetrant PKD1 alleles. These patients illustrate that PKD1 mutations can manifest as a phenocopy of ARPKD with respect to renal involvement and highlight the perils of linkage-based diagnostics in ARPKD without positive PKHD1 mutation data. Furthermore, the phenotypic overlap between ARPKD and these patients resulting from incomplete penetrant PKD1 alleles support a common pathogenesis for these diseases.

Functional analysis of the novel TBX5 c.1333delC mutation resulting in an extended TBX5 protein.

BACKGROUND: Autosomal dominant Holt-Oram syndrome (HOS) is caused by mutations in the TBX5 gene and is characterized by congenital heart and preaxial radial ray upper limb defects. Most of the TBX5 mutations found in patients with HOS cause premature truncation of the primary TBX5 transcript. TBX5 missense mutations alter the three-dimensional structure of the protein and result in failed nuclear localization or reduced binding to target DNA. In this study we present our functional analyses of the novel and unusual c.1333delC mutation found in a patient with classical HOS. METHODS: The functional impact of this novel mutation was assessed by investigating the intracellular localization of the resulting TBX5 protein and its ability to activate the expression of its downstream target ANF. RESULTS: The deletion of the cytosine is the first TBX5 frameshift mutation predicted to result in an elongated TBX5 protein with 74 miscoding amino acids and 62 supernumerary C-terminal amino acids. The c.1333delC mutation affects neither the nuclear localization, nor its colocalization with SALL4, but severely affects the activation of the ANF promoter. CONCLUSION: The mutation c.1333delC does not locate within functional domains, but impairs the activation of the downstream target. This suggests that misfolding of the protein prevents its biological function.

Subtelomeric rearrangements detected in patients with idiopathic mental retardation.

A screening for submicroscopic rearrangements was performed in 111 patients with idiopathic mental retardation (MR) using fluorescence in situ hybridization (FISH) probes from the subtelomeric regions of all chromosome arms. Ten cryptic rearrangements were found (9%): five de novo deletions; one unbalanced de novo translocation; three unbalanced inherited translocations; and one unbalanced recombinant chromosome, inherited from a parent with a pericentric inversion. In addition, 50 of the patients were screened for interstitial rearrangements with spectral karyotyping (SKY), but no aberrations were found. However, SKY detected the subtelomeric rearrangement in three of the four unbalanced translocations. Dysmorphic features were present in all patients with detected subtelomeric rearrangements.

Hereditary multiple and isolated sporadic exostoses in the same kindred: identification of the causative gene (EXT2) and detection of a new mutation, nt112delAT, that distinguishes the two phenotypes.

Hereditary multiple exostoses (HME) is a well known autosomal dominant hereditary orthopedic disorder. Isolated exostoses, on the other hand, occur as sporadic events or as secondary post-traumatic sequel. The occurrence of solitary exostoses in individuals from pedigrees affected with HME may distort conclusions about carrier status and/or diagnosis. Both conditions are potentially malignant and both are associated with genetic alterations in either EXT1 or EXT2 genes. In this study, we present a seven-generation family from western Sweden consisting of 170 blood relatives, 38 of whom had multiple cartilaginous exostoses, while 8 had isolated exostoses. Linkage analysis aimed to discern one of the known EXT genes demonstrated linkage of the HME phenotype to the EXT2 gene. Subsequent mutation analysis revealed a novel mutation, nt112delAT, in this gene. All carriers of the detected mutation had multiple exostoses, indicating full penetrance. None of the pedigree members with isolated exostoses were carriers of the detected mutation. Two of the mutation carriers developed chondrosarcoma yielding a 5.2% risk of malignant development for this mutation. The detection of this mutation has enabled us to provide appropriate genetic counseling concerning this complex situation.

Multiplex ligation-dependent probe amplification (MLPA) detects large deletions in the MECP2 gene of Swedish Rett syndrome patients.

Mutations in the methyl-CpG-binding protein-2 (MECP2) gene on Xq28 have been found to be a cause of Rett syndrome (RS). In a previous mutation screening, we found MECP2 mutations in 81% of Swedish classical Rett women. In this study, we have analyzed 22 patients for MECP2 deletions using multiplex-ligation-dependent probe amplification (MLPA). Clinically, 11 of the patients who were classical Rett women, 3 were forme fruste, 1 was congenital RS, and 7 were Rett variants. As inclusion criteria, we used DNA from patients in whom previous sequencing results showed no mutations in coding portions of the MECP2 gene. MLPA is a method based on multiplex PCR. In one PCR, as many as 40 probes are amplified with the same primers. The specificity of the amplification products is determined by the site-specific hybridization of each probe construct, prior to amplification. Each PCR product has a unique length, which makes it possible to identify it by size separation. In 3 of 11 (27%) classical Rett women, we detected large deletions in MECP2 using MLPA. All these patients had deletions covering two exons; in 2 cases the deletion involved exons 3 and 4 and, in one case, exons 1 and 2 were missing. In the forme fruste, congenital and Rett-variant patients, we found no large deletions. We have found that MLPA is useful when it comes to finding large deletions compromising whole exons in MECP2. Used as a complementary method to DNA sequencing, it revealed new MECP2 mutations in classical RS patients.

Antenatal diagnosis of hereditary fetal growth retardation with aminoaciduria, cholestasis, iron overload, and lactic acidosis in the newborn infant.

OBJECTIVE: A prenatal diagnosis of the fetus for a mother of two previously deceased infants who died from the recently described autosomal recessive disease (OMIM 603358). The infants presented with intrauterine growth retardation, aminoaciduria, cholestasis, iron overload, severe lactic acidosis, and early death (GRACILE syndrome). STUDY DESIGN: DNA was extracted from the fibroblasts and tissue samples of the deceased infants, parental leukocytes, and from a chorion villus biopsy in the next pregnancy. Haplotypes were determined using the relevant markers flanking the disease-associated region of chromosome 2. RESULTS: Both deceased infants were homozygous for the four critical markers. The fetal haploptypes were identical to those of the siblings and the pregnancy was terminated. The iron content of the fetal liver was increased (5000 microg/g) compared with the controls, with a marked iron accumulation in the Kupffer cells. CONCLUSIONS: Antenatal diagnosis can be performed based on linkage analysis in families with at least one affected child because the disease locus has been assigned to a restricted chromosomal region. Typical histological abnormalities may be present in early fetal life.