An accurate diagnosis is an integral component of patient care for children with rare genetic disease. Recent advances in sequencing, in particular whole‐exome sequencing (WES), are identifying the genetic basis of disease for 25–40% of patients. The diagnostic rate is probably influenced by when in the diagnostic process WES is used. The Finding Of Rare Disease GEnes (FORGE) Canada project was a nation‐wide effort to identify mutations for childhood‐onset disorders using WES. Most children enrolled in the FORGE project were toward the end of the diagnostic odyssey. The two primary outcomes of FORGE were novel gene discovery and the identification of mutations in genes known to cause disease. In the latter instance, WES identified mutations in known disease genes for 105 of 362 families studied (29%), thereby informing the impact of WES in the setting of the diagnostic odyssey. Our analysis of this dataset showed that these known disease genes were not identified prior to WES enrollment for two key reasons: genetic heterogeneity associated with a clinical diagnosis and atypical presentation of known, clinically recognized diseases. What is becoming increasingly clear is that WES will be paradigm altering for patients and families with rare genetic diseases.

Hyperekplexia is a human neurological disorder characterized by an excessive startle response and is typically caused by missense and nonsense mutations in the gene encoding the inhibitory glycine receptor (GlyR) α1 subunit (GLRA1) [1][2][3] . Genetic heterogeneity has been confirmed in isolated sporadic cases with mutations in other postsynaptic glycinergic proteins including the GlyR β subunit (GLRB) 4 , gephyrin (GPHN) 5 and RhoGEF collybistin (ARHGEF9) 6 . However, many sporadic patients diagnosed with hyperekplexia do not carry mutations in these genes 2-7 . Here we reveal that missense, nonsense and frameshift mutations in the presynaptic glycine transporter 2 (GlyT2) gene (SLC6A5) 8 also cause hyperekplexia. Patients harbouring mutations in SLC6A5 presented with hypertonia, an exaggerated startle response to tactile or acoustic stimuli, and life-threatening neonatal apnoea episodes. GlyT2 mutations result in defective subcellular localisation and/or decreased glycine uptake, with selected mutations affecting predicted glycine and Na + binding sites. Our results demonstrate that SLC6A5 is a major gene for hyperekplexia and define the first neurological disorder linked to mutations in a Na + /Cl − -dependent transporter for a classical fast neurotransmitter. By analogy, we suggest that in other human disorders where Correspondence and requests for materials (subject to a Material Transfer Agreement) should be addressed to R.J.H. (robert.harvey@pharmacy.ac.uk) or M. I.R. (m.i.rees@swansea.ac.uk).. † these authors contributed equally to this work. COMPETING INTERESTS STATEMENT:The authors declare that they have no competing financial interests. Europe PMC Funders GroupAuthor Manuscript Nat Genet. Author manuscript; available in PMC 2011 October 31. Glycine transporters (GlyTs) are members of the Na + /Cl − -dependent neurotransmitter transporter superfamily 9,10 , integral membrane proteins that utilise electrochemical gradients to control the concentration of neurotransmitters at central synapses. This superfamily also includes transporters for GABA, biogenic amines (norepinephrine, dopamine, serotonin, proline), betaine, taurine and creatine. GlyTs have dual functions at both inhibitory and excitatory synapses, resulting from the differential localisation of two distinct transporters 9,10 , GlyT1 and GlyT2. GlyT1 is predominantly expressed in glial cells 9,10 , exhibits a 2 Na + /1 Cl − /1 glycine stoichiometry and bi-directional glycine transport 11 . These properties are appropriate for the control of extracellular glycine concentrations in the submicromolar range for modulation of N-methyl-D-aspartate receptors 12 , and also for lowering extracellular glycine levels at inhibitory glycinergic synapses 13,14 . By contrast, GlyT2 is found in glycinergic axons, exhibits a 3 Na + /1 Cl − /1 glycine stoichiometry and does not display reverse uptake 11 , reflecting an essential role for GlyT2 in maintaining a high presynaptic pool of neurotransmitter at glycinergic synapses 15 . Na + /Cl − -dependent tr...

Germline mutations in the STK11 gene have been identified in 10-70% of patients with Peutz-Jeghers syndrome (PJS), an autosomal-dominant hamartomatous polyposis syndrome. A second locus was assumed in a large proportion of PJS patients. To date, STK11 alterations comprise mainly point mutations; only a small number of large deletions have been reported. We performed a mutation analysis for the STK11 gene in 71 patients. Of these, 56 met the clinical criteria for PJS and 12 were presumed to have PJS because of mucocutaneous pigmentation only or bowel problems due to isolated PJS polyps. No clinical information was available for the remaining three patients. By direct sequencing of the coding region of the STK11 gene, we identified point mutations in 37 of 71 patients (52%). We examined the remaining 34 patients by means of the multiplex ligation-dependent probe amplification (MLPA) method, and detected deletions in 17 patients. In four patients the deletion extended over all 10 exons, and in eight patients only the promoter region and exon 1 were deleted. The remaining deletions encompassed exons 2-10 (in two patients), exons 2-3, exons 4-5, or exon 8. When only patients who met the clinical criteria for PJS are considered, the overall mutation detection rate increases to 94% (64% point mutations and 30% large deletions). No mutation was identified in any of the 12 presumed cases. In conclusion, we found that approximately one-third of the patients who met the clinical PJS criteria exhibited large genomic deletions that were readily detectable by MLPA. Screening for point mutations and large deletions by direct sequencing or MLPA, respectively, increased the mutation detection rate in the STK11 gene up to 94%. There may be still other mutations in the STK11 gene that are not detectable by the methods applied here. Therefore, it is questionable whether a second PJS locus exists at all.

Holt-Oram syndrome (HOS) is an autosomal dominant hearthand syndrome characterized by congenital heart disease (CHD) and upper limb deformity, and caused by mutations in the TBX5 gene. To date, the sensitivity of TBX5 genetic testing for HOS has been unclear. We now report mutational analyses of a nongenetically selected population of 54 unrelated individuals who were consecutively referred to our center with a clinical diagnosis of HOS. TBX5 mutational analyses were performed in all individuals, and clinical histories and findings were reviewed for each patient without reference to the genotypes. Twenty-six percent of the complete cohort was shown to have mutations of the TBX5 gene. However, among those subjects for whom clinical review demonstrated that their presentations met strict diagnostic criteria for HOS, TBX5 mutations were identified in 74%. No mutations were identified in those subjects who did not meet these criteria. Thus, these studies validate our clinical diagnostic criteria for HOS including an absolute requirement for preaxial radial ray upper limb malformation. Accordingly, TBX5 genotyping has high sensitivity and specificity for HOS if stringent diagnostic criteria are used in assigning the clinical diagnosis. HOS is the most common of the heart-hand syndromes. It segregates in an autosomal dominant fashion and is estimated to occur in at least 1/100,000 live births (1). HOS is characterized by upper limb anomalies involving the preaxial radial ray and CHD (2,3). Upper limb deformity may be bilateral but asymmetric or even unilateral. The most common forms of CHD associated with HOS are ASD, usually of the ostium secundum variety, and VSD, usually occurring in the muscular trabeculated septum. Cardiac conduction disease may also occur, regardless of the presence or absence of structural cardiac disease (3

This study reports the clinical course of two patients with osteogenesis imperfecta who received prenatal human fetal mesenchymal stem cell (MSC) transplantation and postnatal boosting with same‐donor MSCs. Findings suggest that prenatal transplantation of allogeneic human fetal MSCs in osteogenesis imperfecta appears safe and is of likely clinical benefit and that retransplantation with same‐donor cells is feasible. Further studies are required.

Congenital insensitivity to pain with anhidrosis (CIPA) is characterized by recurrent episodes of unexplained fever, anhidrosis (inability to sweat), absence of reaction to noxious stimuli, self-mutilating behavior, and mental retardation. Human TRKA encodes a high-affinity tyrosine kinase receptor for nerve growth factor (NGF), a member of the neurotrophin family that induces neurite outgrowth and promotes survival of embryonic sensory and sympathetic neurons. We have recently demonstrated that TRKA is responsible for CIPA by identifying three mutations in a region encoding the intracellular tyrosine kinase domain of TRKA in one Ecuadorian and three Japanese families. We have developed a comprehensive strategy to screen for TRKA mutations, on the basis of the gene's structure and organization. Here we report 11 novel mutations, in seven affected families. These are six missense mutations, two frameshift mutations, one nonsense mutation, and two splice-site mutations. Mendelian inheritance of the mutations is confirmed in six families for which parent samples are available. Two mutations are linked, on the same chromosome, to Arg85Ser and to His598Tyr;Gly607Val, hence, they probably represent double and triple mutations. The mutations are distributed in an extracellular domain, involved in NGF binding, as well as the intracellular signal-transduction domain. These data suggest that TRKA defects cause CIPA in various ethnic groups.

Context: Beckwith-Wiedemann syndrome (BWS) arises by several genetic and epigenetic mechanisms affecting the balance of imprinted gene expression in chromosome 11p15.5. The most frequent alteration associated with BWS is the absence of methylation at the maternal allele of KvDMR1, an intronic CpG island within the KCNQ1 gene. Targeted deletion of KvDMR1 suggests that this locus is an imprinting control region (ICR) that regulates multiple genes in 11p15.5. Cell culture based enhancer blocking assays indicate that KvDMR1 may function as a methylation modulated chromatin insulator and/or silencer. Objective: To determine the potential consequence of loss of methylation (LOM) at KvDMR1 in the development of BWS. Methods: The steady state levels of CDKN1C gene expression in fibroblast cells from normal individuals, and from persons with BWS who have LOM at KvDMR1, was determined by both real time quantitative polymerase chain reaction (qPCR) and ribonuclease protection assay (RPA). Methylation of the CDKN1C promoter region was assessed by Southern hybridisation using a methylation sensitive restriction endonuclease. Results: Both qPCR and RPA clearly demonstrated a marked decrease (86-93%) in the expression level of the CDKN1C gene in cells derived from patients with BWS, who had LOM at KvDMR1. Southern analysis indicated that downregulation of CDKN1C in these patients was not associated with hypermethylation at the presumptive CDKN1C promoter. Conclusions: An epimutation at KvDMR1, the absence of maternal methylation, causes the aberrant silencing of CDKN1C, some 180 kb away on the maternal chromosome. Similar to mutations at this locus, this silencing may give rise to BWS.

PurposeThe purpose of the current study was to assess the penetrance of NRXN1 deletions.MethodsWe compared the prevalence and genomic extent of NRXN1 deletions identified among 19,263 clinically referred cases to that of 15,264 controls. The burden of additional clinically relevant CNVs was used as a proxy to estimate the relative penetrance of NRXN1 deletions.ResultsWe identified 41 (0.21%) previously unreported exonic NRXN1 deletions ascertained for developmental delay/intellectual disability, significantly greater than in controls [OR=8.14 (95% CI 2.91–22.72), p< 0.0001)]. Ten (22.7%) of these had a second clinically relevant CNV. Subjects with a deletion near the 3′ end of NRXN1 were significantly more likely to have a second rare CNV than subjects with a 5′ NRXN1 deletion [OR=7.47 (95% CI 2.36–23.61), p=0.0006]. The prevalence of intronic NRXN1 deletions was not statistically different between cases and controls (p=0.618). The majority (63.2%) of intronic NRXN1 deletion cases had a second rare CNV, a two-fold greater prevalence than for exonic NRXN1 deletion cases (p=0.0035).ConclusionsThe results support the importance of exons near the 5′ end of NRXN1 in the expression of neurodevelopmental disorders. Intronic NRXN1 deletions do not appear to substantially increase the risk for clinical phenotypes.