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Collier/Olf/EBF (COE) transcription factors have distinct expression patterns in the developing and mature nervous system. To date, a neurological disease association has been conclusively established for only the Early B-cell Factor-3 (EBF3) COE family member through the identification of heterozygous loss-of-function variants in individuals with autism spectrum/neurodevelopmental disorders (NDD). Here, we identify a symptom severity risk association with missense variants primarily disrupting the zinc finger domain (ZNF) in EBF3-related NDD.A phenotypic assessment of 41 individuals was combined with a literature meta-analysis for a total of 83 individuals diagnosed with EBF3-related NDD. Quantitative diagnostic phenotypic and symptom severity scales were developed to compare EBF3 variant type and location to identify genotype-phenotype correlations. To stratify the effects of EBF3 variants disrupting either the DNA-binding domain (DBD) or the ZNF, we used in vivo fruit fly UAS-GAL4 expression and in vitro luciferase assays.We show that patient symptom severity correlates with EBF3 missense variants perturbing the ZNF, which is a key protein domain required for stabilizing the interaction between EBF3 and the target DNA sequence. We found that ZNF-associated variants failed to restore viability in the fruit fly and impaired transcriptional activation. However, the recurrent variant EBF3 p.Arg209Trp in the DBD is capable of partially rescuing viability in the fly and preserved transcriptional activation.We describe a symptom severity risk association with ZNF perturbations and EBF3 loss-of-function in the largest reported cohort to date of EBF3-related NDD patients. This analysis should have potential predictive clinical value for newly identified patients with EBF3 gene variants. ANN NEUROL 2022;92:138-153.

Purpose Growth differentiation factor 11 (GDF11) is a key signaling protein required for proper development of many organ systems. Only one prior study has associated an inherited GDF11 variant with a dominant human disease in a family with variable craniofacial and vertebral abnormalities. Here, we expand the phenotypic spectrum associated with GDF11 variants and document the nature of the variants. Methods We present a cohort of six probands with de novo and inherited nonsense/frameshift (4/6 patients) and missense (2/6) variants in GDF11. We generated gdf11 mutant zebrafish to model loss of gdf11 phenotypes and used an overexpression screen in Drosophila to test variant functionality. Results Patients with variants in GDF11 presented with craniofacial (5/6) , vertebral (5/6), neurological (6/6), visual (4/6), cardiac (3/6), auditory (3/6) and connective tissue abnormalities (3/6). gdf11 mutant zebrafish show craniofacial abnormalities and body segmentation defects that match some patient phenotypes. Expression of the patients’ variants in the fly showed that one nonsense variant in GDF11 is a severe loss-of-function (LOF) alleles whereas the missense variants in our cohort are partial LOF variants. Conclusion GDF11 is needed for human development, particularly neuronal development, and LOF GDF11 alleles can affect the development of numerous organs and tissues.

Oligodontia is the congenital absence of six or more teeth and comprises the more severe forms of tooth agenesis. Many genes have been implicated in the etiology of tooth agenesis, which is highly variable in its clinical presentation. The purpose of this study was to identify associations between genetic mutations and clinical features of oligodontia patients. An online systematic search of papers published from January 1992 to June 2021 identified 381 oligodontia cases meeting the eligibility criteria of causative gene mutation, phenotype description, and radiographic records. Additionally, ten families with oligodontia were recruited and their genetic etiologies were determined by whole-exome sequence analyses. We identified a novel mutation in WNT10A (c.99_105dup) and eight previously reported mutations in WNT10A (c.433 G > A; c.682 T > A; c.318 C > G; c.511.C > T; c.321 C > A), EDAR (c.581 C > T), and LRP6 (c.1003 C > T, c.2747 G > T). Collectively, 20 different causative genes were implicated among those 393 cases with oligodontia. For each causative gene, the mean number of missing teeth per case and the frequency of teeth missing at each position were calculated. Genotype–phenotype correlation analysis indicated that molars agenesis is more likely linked to PAX9 mutations, mandibular first premolar agenesis is least associated with PAX9 mutations. Mandibular incisors and maxillary lateral incisor agenesis are most closely linked to EDA mutations.

The phenotypic variability associated with pathogenic variants in Lysine Acetyltransferase 6B (KAT6B, a.k.a. MORF, MYST4) results in several interrelated syndromes including Say‐Barber‐Biesecker‐Young‐Simpson Syndrome and Genitopatellar Syndrome. Here we present 20 new cases representing 10 novel KAT6B variants. These patients exhibit a range of clinical phenotypes including intellectual disability, mobility and language difficulties, craniofacial dysmorphology, and skeletal anomalies. Given the range of features previously described for KAT6B‐related syndromes, we have identified additional phenotypes including concern for keratoconus, sensitivity to light or noise, recurring infections, and fractures in greater numbers than previously reported. We surveyed clinicians to qualitatively assess the ways families engage with genetic counselors upon diagnosis. We found that 56% (10/18) of individuals receive diagnoses before the age of 2 years (median age = 1.96 years), making it challenging to address future complications with limited accessible information and vast phenotypic severity. We used CRISPR to introduce truncating variants into the KAT6B gene in model cell lines and performed chromatin accessibility and transcriptome sequencing to identify key dysregulated pathways. This study expands the clinical spectrum and addresses the challenges to management and genetic counseling for patients with KAT6B‐related disorders., We describe 20 new cases harboring the KAT6B spectrum of disorders, which range from Say‐Barber‐Biesecker‐Young‐Simpson (SBBYSS) to Genitopatellar (GPS) syndrome or an intermediate phenotype. In our holistic approach, we expand the genotypic and phenotypic spectrum of KAT6B spectrum of disorders. Furthermore, we provide extensive clinical phenotyping, explore the impact of genetic counseling for these complex syndromes, and examine molecular mechanisms in RNA‐seq data in an in vitro cell model of truncating KAT6B mutations.

Hypotonia, Ataxia, and Delayed Development syndrome is a neurodevelopmental disorder caused by heterozygousEarly B-Cell Factor 3(EBF3) loss-of-function variants. Identified in 2016, the full spectrum of clinical findings and the relationship between theEBF3genotype and clinical outcomes has not been determined beyond its namesake features. We combined a phenotypic assessment of 33 individuals molecularly diagnosed withEBF3pathogenic variants with a meta-analysis of 34 previously reported individuals. The combined 62 unique individuals enabled comparative cross-sectional phenotype and genotype analysis in the largest cohort to date of affected individuals. Cardinal distinguishing features were identified that facilitate phenotypic stratification for clinical diagnosis. We developed assessment scales to ascertain individuals at risk for pathogenicEBF3variants, stratify the clinical severity, and connect variant-specific molecular phenotypes to clinical outcomes. Our findings show that a specific class ofEBF3variants affecting the evolutionarily conserved Zinc Finger (ZNF) motif, which is critical for stabilizing the protein interaction with the DNA target sequence, is associated with an increased risk of persistent motor and language impairments. These findings highlight the impact of combining variant-specific molecular phenotypes with comprehensive clinical data to predict neurodevelopmental outcomes and potentially guide personalized decisions for therapeutic interventions.

BackgroundWe aimed to define the clinical and mutational spectrum, and to provide novel molecular insights into DHX30-associated neurodevelopmental disorder.MethodsClinical and genetic data from affected individuals were collected through family support group, GeneMatcher and our network of collaborators. We investigated the impact of novel missense variants with respect to ATPase and helicase activity, stress granule (SG) formation, global translation, and their effect on embryonic development in zebrafish. SG formation was additionally analyzed in CRISPR/Cas9-mediated DHX30-deficient HEK293T and zebrafish models, along with in vivo behavioral assays.ResultsWe identified 25 previously unreported individuals, ten of whom carry novel variants, two of which are recurrent, and provide evidence of gonadal mosaicism in one family. All 19 individuals harboring heterozygous missense variants within helicase core motifs (HCMs) have global developmental delay, intellectual disability, severe speech impairment and gait abnormalities. These variants impair the ATPase and helicase activity of DHX30, trigger SG formation, interfere with global translation, and cause developmental defects in a zebrafish model. Notably, 4 individuals harboring heterozygous variants resulting either in haploinsufficiency or truncated proteins presented with a milder clinical course, similar to an individual bearing a de novo mosaic HCM missense variant. Functionally, we established DHX30 as an ATP-dependent RNA helicase and as an evolutionary conserved factor in SG assembly. Based on the clinical course, the variant location and type we establish two distinct clinical subtypes. DHX30 loss-of-function mutations cause a milder phenotype whereas a severe phenotype is caused by HCM missense mutations that, in addition to the loss of ATPase and helicase activity, lead to a detrimental gain-of function with respect to SG formation. Behavioral characterization of dhx30 deficient zebrafish revealed altered sleep-wake activity and social interaction, partially resembling the human phenotype.ConclusionsOur study highlights the usefulness of social media in order to define novel Mendelian disorders, and exemplifies how functional analyses accompanied by clinical and genetic findings can define clinically distinct subtypes for ultra-rare disorders. Such approaches require close interdisciplinary collaboration between families/legal representatives of the affected individuals, clinicians, molecular genetics diagnostic laboratories and research laboratories.

Primary mitochondrial complex I deficiency is the most common defect of the mitochondrial respiratory chain. It is caused by defects in structural components and assembly factors of this large protein complex. Mutations in the assembly factor NDUFAF5 are rare, with only five families reported to date. This study provides clinical, biochemical, molecular and functional data for four unrelated additional families, and three novel pathogenic variants. Three cases presented in infancy with lactic acidosis and classic Leigh syndrome. One patient, however, has a milder phenotype, with symptoms starting at 27 months and a protracted clinical course with improvement and relapsing episodes. She is homozygous for a previously reported mutation, p.Met279Arg and alive at 19 years with mild neurological involvement, normal lactate but abnormal urine organic acids. We found the same mutation in one of our severely affected patients in compound heterozygosity with a novel p.Lys52Thr mutation. Both patients with p.Met279Arg are of Taiwanese descent and had severe hyponatremia. Our third and fourth patients, both Caucasian, shared a common, newly described, missense mutation p.Lys109Asn which we show induces skipping of exon 3. Both Caucasian patients were compound heterozygotes, one with a previously reported Ashkenazi founder mutation while the other was negative for additional exonic variants. Whole genome sequencing followed by RNA studies revealed a novel deep intronic variant at position c.223-907A>C inducing an exonic splice enhancer. Our report adds significant new information to the mutational spectrum of NDUFAF5, further delineating the phenotypic heterogeneity of this mitochondrial defect.

Background Patients with autism spectrum disorder and developmental delay or encephalopathy rarely demonstrate no or negligible hair and nail growth, suggesting a biotin-responsive clinical disorder. Methods A ten-year-old girl presented with features of autism spectrum disorder, isolated headaches, and episodes of headaches and limb shaking. Her medical history revealed that her hair and nails did not grow. Administration of biotin restored her nail and hair growth and improved intellectual ability and school performance. Her episodes of headaches, single limb shaking, and loss of consciousness responded to administration of acetazolamide, and her school performance and social skills further improved. Results A de novo c.1091 C > T, p.T364M pathogenic variant was found in the ATP1A2 gene by whole-exome sequencing, but a genetic etiology in the biotin-responsive metabolic pathways was not identified. Conclusions The combination of biotin and acetazolamide treatment was successful in restoring normal mental function and school performance. Poor or no clinical nail and hair growth in any child with a developmental delay-autism spectrum disorder presentation should be considered as evidence for a biotin-responsive genetic disorder even when exome testing is negative.

Reanalysis of Clinical Exome Data and Diagnostic Yield As knowledge about genetic causes of disease improves, periodic reanalysis of clinical exome sequence could yield new genetic information. Thi...

BackgroundThe list of Mendelian disorders of the epigenetic machinery has expanded rapidly during the last 5 years. A few missense variants in the chromatin remodeler CHD1 have been found in several large-scale sequencing efforts focused on uncovering the genetic aetiology of autism.ObjectivesTo explore whether variants in CHD1 are associated with a human phenotype.MethodsWe used GeneMatcher to identify other physicians caring for patients with variants in CHD1. We also explored the epigenetic consequences of one of these variants in cultured fibroblasts.ResultsHere we describe six CHD1 heterozygous missense variants in a cohort of patients with autism, speech apraxia, developmental delay and facial dysmorphic features. Importantly, three of these variants occurred de novo. We also report on a subject with a de novo deletion covering a large fraction of the CHD1 gene without any obvious neurological phenotype. Finally, we demonstrate increased levels of the closed chromatin modification H3K27me3 in fibroblasts from a subject carrying a de novo variant in CHD1.ConclusionsOur results suggest that variants in CHD1 can lead to diverse phenotypic outcomes; however, the neurodevelopmental phenotype appears to be limited to patients with missense variants, which is compatible with a dominant negative mechanism of disease.

Membrane Protein Palmitoylated 5 (MPP5) is a highly conserved apical complex protein essential for cell polarity, fate and survival. Defects in cell polarity are associated with neurologic disorders including autism and microcephaly. MPP5 is essential for neurogenesis in animal models, but human variants leading to neurologic impairment have not been described. We identified three patients with heterozygous MPP5 de novo variants (DNV) and global developmental delay (GDD) and compared their phenotypes and magnetic resonance imaging (MRI) to ascertain how MPP5 DNV leads to GDD. All three patients with MPP5 DNV experienced GDD with language delay/regression and behavioral changes. MRI ranged from normal to decreased gyral folding and microcephaly. The effects of MPP5 depletion on the developing brain were assessed by creating a heterozygous conditional knock out (het CKO) murine model with central nervous system (CNS)-specific Nestin-Cre drivers. In the het CKO model, Mpp5 depletion led to microcephaly, decreased cerebellar volume and cortical thickness. Het CKO mice had decreased ependymal cells and Mpp5 at the apical surface of cortical ventricular zone compared with wild type. Het CKO mice also failed to maintain progenitor pools essential for neurogenesis. The proportion of cortical cells undergoing apoptotic cell death increased, suggesting that cell death reduces progenitor population and neuron number. Het CKO mice also showed behavioral changes, similar to our patients. To our knowledge, this is the first report to show that variants in MPP5 are associated with GDD, behavioral abnormalities and language regression/delay. Murine modeling shows that neurogenesis is likely altered in these individuals, with cell death and skewed cellular composition playing significant roles.

The human sodium-dependent multivitamin transporter (hSMVT) is a product of the SLC5A6 gene and mediates biotin, pantothenic acid, and lipoate uptake in a variety of cellular systems. We report here the identification of mutations R94X, a premature termination, and R123L, a dysfunctional amino acid change, both in exon 3 of the SLC5A6 gene in a child using whole genome-scanning. At 15 months of age, the child showed failure to thrive, microcephaly and brain changes on MRI, cerebral palsy and developmental delay, variable immunodeficiency, and severe gastro-esophageal reflux requiring a gastrostomy tube/fundoplication, osteoporosis, and pathologic bone fractures. After identification of the SLC5A6 mutations, he responded clinically to supplemental administration of excess biotin, pantothenic acid, and lipoate with improvement in clinical findings. Functionality of the two mutants was examined by 3H-biotin uptake assay following expression of the mutants in human-derived intestinal HuTu-80 and brain U87 cells. The results showed severe impairment in biotin uptake in cells expressing the mutants compared to those expressing wild-type hSMVT. Live cell confocal imaging of cells expressing the mutants showed the R94X mutant to be poorly tolerated and localized in the cytoplasm, while the R123L mutant was predominantly retained in the endoplasmic reticulum. This is the first reporting of mutations in the SLC5A6 gene in man, and suggests that this gene is important for brain development and a wide variety of clinical functions.

Serine biosynthesis defects are autosomal recessive metabolic disorders resulting from the deficiency of any of the three enzymes involved in de novo serine biosynthesis, specifically phosphoglycerate dehydrogenase (PGDH), phosphoserine aminotransferase (PSAT), and phosphoserine phosphatase (PSP). In this study, we performed metabolomic profiling on 4 children with serine biosynthesis defects; 3 with PGDH deficiency and 1 with PSAT deficiency. The evaluations were performed at baseline and with serine and glycine supplementation. Metabolomic profiling performed at baseline showed low phospholipid species, including glycerophosphocholine, glycerophosphoethanolamine, and sphingomyelin. All children had low serine and glycine as expected. Low glycerophosphocholine compounds were found in 4 children, low glycerophosphoethanolamine compounds in 3 children, and low sphingomyelin species in 2 children. Metabolic profiling with serine and glycine supplementation showed normalization of most of the low phospholipid compounds in the 4 children. Phospholipids are the major component of plasma and intracellular membranes, and phosphatidylcholine is the most abundant phospholipid of all mammalian cell types and subcellular organelles. Phosphatidylcholine is of particular importance for the nervous system, where it is essential for neuronal differentiation. The observed low phosphatidylcholine species in children with serine biosynthesis defects that improved after serine supplementation, supports the role of serine as a significant precursor for phosphatidylcholine. The vital role that phosphatidylcholine has during neuronal differentiation and the pronounced neurological manifestations in serine biosynthesis defects suggest that phosphatidylcholine deficiency occurring secondary to serine deficiency may have a significant contribution to the development of the neurological manifestations in individuals with serine biosynthesis defects.

The list of Mendelian disorders of the epigenetic machinery has expanded rapidly during the last five years. A few missense variants in the chromatin remodeler CHD1 have been found in several large scale sequencing efforts focused on uncovering the genetic etiology of autism. Here we describe CHD1 heterozygous missense variants in a cohort of patients with autism, speech apraxia, developmental delay and facial dysmorphic features. Importantly three of these variants occurred de novo. We also report on a patient with a de novo deletion covering a large fraction of the CHD1 gene without any obvious neurological phenotype. Our results suggest that variants in CHD1 can lead to diverse phenotypic outcomes; however, the neurodevelopmental phenotype appears to be limited to patients with missense variants, which is compatible with a dominant negative mechanism of disease.

Serine biosynthesis defects can present in a broad phenotypic spectrum ranging from Neu-Laxova syndrome, a lethal disease with multiple congenital anomalies at the severe end, to an infantile disease with severe psychomotor retardation and seizures as an intermediate phenotype, to a childhood disease with intellectual disability at the mild end. In this report we present 6 individuals from 3 families with infantile phosphoglycerate dehydrogenase (PGDH) deficiency who presented with psychomotor delay, growth failure, microcephaly, and spasticity. The phenotype was variable with absence of seizures in 2 sisters in family 1 and 1 infant in family 2 and seizures with pronounced happy affect in 3 sisters in family 3. The initiation of serine treatment had pronounced effect on seizures and spasticity in the sisters in family 3, but minimal developmental effects on the children in families 1 and 2. With such phenotypic variability, the diagnosis of PGDH deficiency can be challenging.

Item does not contain fulltext Developmental and epileptic encephalopathy (DEE) is a group of conditions characterized by the co-occurrence of epilepsy and intellectual disability (ID), typically with developmental plateauing or regression associated with frequent epileptiform activity. The cause of DEE remains unknown in the majority of cases. We performed whole-genome sequencing (WGS) in 197 individuals with unexplained DEE and pharmaco-resistant seizures and in their unaffected parents. We focused our attention on de novo mutations (DNMs) and identified candidate genes containing such variants. We sought to identify additional subjects with DNMs in these genes by performing targeted sequencing in another series of individuals with DEE and by mining various sequencing datasets. We also performed meta-analyses to document enrichment of DNMs in candidate genes by leveraging our WGS dataset with those of several DEE and ID series. By combining these strategies, we were able to provide a causal link between DEE and the following genes: NTRK2, GABRB2, CLTC, DHDDS, NUS1, RAB11A, GABBR2, and SNAP25. Overall, we established a molecular diagnosis in 63/197 (32%) individuals in our WGS series. The main cause of DEE in these individuals was de novo point mutations (53/63 solved cases), followed by inherited mutations (6/63 solved cases) and de novo CNVs (4/63 solved cases). De novo missense variants explained a larger proportion of individuals in our series than in other series that were primarily ascertained because of ID. Moreover, these DNMs were more frequently recurrent than those identified in ID series. These observations indicate that the genetic landscape of DEE might be different from that of ID without epilepsy.

Diagnosis of genetic syndromes may be difficult when specific components of a disorder manifest at a later age. We present a follow up of a previous report [Seeherunvong et al., (2004); AJMGA 127: 149-151], of an individual with 22q duplication and sex-reversal syndrome. The subject's phenotype evolved to include peripheral and central demyelination, Waardenburg syndrome type IV, and Hirschsprung disease (PCWH; MIM 609136). DNA microarray analysis defined the duplication at 22q11.2q13, including SOX10. Sequencing of the coding region of SOX10 did not reveal any mutations. Our data suggest that SOX10 duplication can cause disorders of sex development and PCWH, supporting the hypothesis that SOX10 toxic gain of function rather than dominant negative activity underlies PCWH.

The X-linked dominant trait focal dermal hypoplasia (FDH, Goltz syndrome) is a developmental defect with focal distribution of affected tissues due to a block of Wnt signal transmission from cells carrying a detrimental PORCN mutation on an active X-chromosome. Molecular characterization of 24 unrelated patients from different ethnic backgrounds revealed 23 different mutations of the PORCN gene in Xp11.23. Three were microdeletions eliminating PORCN and encompassing neighboring genes such as EBP, the gene associated with Conradi-Hunermann-Happle syndrome (CDPX2). 12/24 patients carried nonsense mutations resulting in loss of function. In one case a canonical splice acceptor site was mutated, and 8 missense mutations exchanged highly conserved amino acids. FDH patients overcome the consequences of potentially lethal X-chromosomal mutations by extreme skewing of X-chromosome inactivation in females, enabling transmission of the trait in families, or by postzygotic mosaicism both in male and female individuals. Molecular characterization of the PORCN mutations in cases diagnosed as Goltz syndrome is particularly relevant for genetic counseling of patients and their families since no functional diagnostic test is available and carriers of the mutation might otherwise be overlooked due to considerable phenotypic variability associated with the mosaic status.

Gain-of-function mutations of NaV1.7 have been shown to produce two distinct disorders: NaV1.7 mutations that enhance activation produce inherited erythromelalgia (IEM), characterized by burning pain in the extremities; NaV1.7 mutations that impair inactivation produce a different, nonoverlapping syndrome, paroxysmal extreme pain disorder (PEPD), characterized by rectal, periocular, and perimandibular pain. Here we report a novel NaV1.7 mutation associated with a mixed clinical phenotype with characteristics of IEM and PEPD, with an alanine 1632 substitution by glutamate (A1632E) in domain IV S4–S5 linker. Patch-clamp analysis shows that A1632E produces changes in channel function seen in both IEM and PEPD mutations: A1632E hyperpolarizes (−7 mV) the voltage dependence of activation, slows deactivation, and enhances ramp responses, as observed in NaV1.7 mutations that produce IEM. A1632E depolarizes (+17mV) the voltage dependence of fast inactivation, slows fast inactivation, and prevents full inactivation, resulting in persistent inward currents similar to PEPD mutations. Using current clamp, we show that A1632E renders dorsal root ganglion (DRG) and trigeminal ganglion neurons hyperexcitable. These results demonstrate a NaV1.7 mutant with biophysical characteristics common to PEPD (impaired fast inactivation) and IEM (hyperpolarized activation, slow deactivation, and enhanced ramp currents) associated with a clinical phenotype with characteristics of both IEM and PEPD and show that this mutation renders DRG and trigeminal ganglion neurons hyperexcitable. These observations indicate that IEM and PEPD mutants are part of a physiological continuum that can produce a continuum of clinical phenotypes.

Purpose: Long QT Syndrome, Marfan Syndrome, hypertrophic and dilated cardiomyopathy are caused by mutations in large, multi-exon genes that are principally expressed in cardiovascular tissues. Genetic testing for these disorders is labor-intensive and expensive. We sought to develop a more rapid, comprehensive, and cost-effective approach. Methods: Paired whole blood samples were collected into tubes with or without an RNA-preserving solution, and harvested for whole blood RNA or leukocyte DNA, respectively. Large overlapping cDNA fragments from KCNQ1 and KCNH2 (Long QT Syndrome), MYBPC3 (hypertrophic and dilated cardiomyopathy), or FBN1 (Marfan Syndrome) were amplified from RNA and directly sequenced. Variants were confirmed in leukocyte DNA. Results: All 4 transcripts were amplified and sequenced from whole blood mRNA. Six known and 2 novel mutations were first identified from RNA of 10 probands, and later confirmed in genomic DNA, at considerable savings in time and cost. In one patient with MFS, RNA sequencing directly identified a splicing mutation. Results from RNA and DNA were concordant for single nucleotide polymorphisms at the same loci. Conclusion: Taking advantage of new whole blood RNA stabilization methods, we have designed a cost-effective, comprehensive method for mutation detection that should significantly facilitate clinical genetic testing in four lethal cardiovascular disorders.

Supernumerary marker chromosomes (SMCs) without detectable alphoid DNA are predicted to have a neocentromere and have been referred to as mitotically stable neocentromere marker chromosomes (NMCs). We report the molecular cytogenetic characterization of a new case with analphoid NMC derived from 15q25-->qter using high-resolution comparative genomic hybridization (HR-CGH) and multiplex fluorescence in situ hybridization analyses with various alpha-satellite DNA probes, all-human-centromere probe (AHC), whole chromosome painting probes, and a subtelomere probe. The propositus is a dysmorphic infant who, at age 3 months, showed accelerated growth, partial deafness, and a phenotype similar to that of the eight previously reported cases of distal 15q tetrasomy. Chromosome studies showed that he had a de novo extra SMC in 80% of cells examined. HR-CGH revealed rev ish enh(15)(q25qter). Molecular cytogenetic analysis and molecular DNA polymorphism study demonstrated that this extra SMC is an NMC containing an inverted duplication of the distal long arm of chromosome 15 (tetrasomy 15q25-->qter) which originated paternally, i.e. ish der(15)(qte-->q25::q25[neocen]-->qter)(AHC-, CEP15-, WCP15+, PCP15q++). This case further elucidates the phenotype related to tetrasomy of this specific chromosome segment and represents a new report of a neocentromere on distal chromosome 15q suggesting that this region appears to be susceptible to the formation of neocentromeres.

Cayman ataxia is a recessive congenital ataxia restricted to one area of Grand Cayman Island. Comparative mapping suggested that the locus on 19p13.3 associated with Cayman ataxia might be homologous to the locus on mouse chromosome 10 associated with the recessive ataxic mouse mutant jittery. Screening genes in the region of overlap identified mutations in a novel predicted gene in three mouse jittery alleles, including the first mouse mutation caused by an Alu-related (B1 element) insertion. We found two mutations exclusively in all individuals with Cayman ataxia. The gene ATCAY or Atcay encodes a neuron-restricted protein called caytaxin. Caytaxin contains a CRAL-TRIO motif common to proteins that bind small lipophilic molecules. Mutations in another protein containing a CRAL-TRIO domain, alpha-tocopherol transfer protein (TTPA), cause a vitamin E-responsive ataxia. Three-dimensional protein structural modeling predicts that the caytaxin ligand is more polar than vitamin E. Identification of the caytaxin ligand may help develop a therapy for Cayman ataxia.

WNT10A is a signaling molecule involved in tooth development, and WNT10A defects are associated with tooth agenesis. We characterized Wnt10a null mice generated by the knockout mouse project (KOMP) and six families with WNT10A mutations, including a novel p.Arg104Cys defect, in the absence of EDA,EDAR, or EDARADD variations. Wnt10a null mice exhibited supernumerary mandibular fourth molars, and smaller molars with abnormal cusp patterning and root taurodontism. Wnt10a (-/-) incisors showed distinctive apical-lingual wedge-shaped defects. These findings spurred us to closely examine the dental phenotypes of our WNT10A families. WNT10A heterozygotes exhibited molar root taurodontism and mild tooth agenesis (with incomplete penetrance) in their permanent dentitions. Individuals with two defective WNT10A alleles showed severe tooth agenesis and had fewer cusps on their molars. The misshapened molar crowns and roots were consistent with the Wnt10a null phenotype and were not previously associated with WNT10A defects. The missing teeth contrasted with the presence of supplemental teeth in the Wnt10a null mice and demonstrated mammalian species differences in the roles of Wnt signaling in early tooth development. We conclude that molar crown and root dysmorphologies are caused by WNT10A defects and that the severity of the tooth agenesis correlates with the number of defective WNT10A alleles.

We have examined the transcript levels of a variety of oxidative phosphorylation (OXPHOS) and associated bioenergetic genes in tissues of a patient carrying the myopathy, encephalopathy, lactic acidosis, and stroke-like episodes (MELAS) A3243G mitochondrial DNA (mtDNA) mutation and the skeletal muscles of 14 patients harboring other pathogenic mtDNA mutations. The patients' tissues, which harbored 88% or more mutant mtDNA, had increased levels of mtDNA transcripts, increased nuclear OXPHOS gene transcripts including the ATP synthase beta subunit and the heart-muscle isoform of the adenine nucleotide translocator, and increased ancillary gene transcripts including muscle mitochondrial creatine phosphokinase, muscle glycogen phosphorylase, hexokinase I, muscle phosphofructokinase, the E1alpha subunit of pyruvate dehydrogenase, and the ubiquinone oxidoreductase. A similar coordinate induction of bioenergetic genes was observed in the muscle biopsies of severe pathologic mtDNA mutations. The more significant coordinated expression was found in muscle from patients with the MELAS, myoclonic epilepsy with ragged red fibers, and chronic progressive external ophthalmoplegia deletion syndromes, with ragged red muscle fibers and mitochondrial paracrystalline inclusions. High levels of mutant mtDNAs were linked to a high induction of the mtDNA and nuclear OXPHOS genes and of several associated bioenergetic genes. These observations suggest that human tissues attempt to compensate for OXPHOS defects associated with mtDNA mutations by stimulating mitochondrial biogenesis, possibly mediated through redox-sensitive transcription factors.

An anterior twin with trisomy 21 at the time of amniocentesis was posterior at the termination of the pregnancy. Reversal of position must be considered in twin fetuses discordant for a genetic disorder.

A recent study found a high prevalence of a missense mutation (S135L) in the gene for galactose 1-phosphate uridyltransferase (GALT) in black children with galactosemia (J Pediatr 1996;128:89-95). In the present study, GALT activity and GALT protein content were measured in erythrocytes and leukocytes of eight black and seven white galactosemic (GALT-deficient) children, for correlation with the presence of the S135L and Q188R (highly prevalent in white galactosemic children) missense mutations. The S135L mutation was found in 9 of 16 alleles of black children but not in white children; the Q188R mutation was found in 10 of 14 alleles examined in white galactosemic children and in 4 of 16 alleles in black galactosemic children. The GALT activity was near zero in the erythrocytes of white and black galactosemic children (0.26 ± 0.28 vs 0.33 ± 0.25 μmol/hr per gram of hemoglobin, respectively; p = 0.61) (normal 17 to 26 μmol/hr per gram), and no correlation of erythrocyte activity with genotype was observed. The GALT activity was higher in the leukocytes of black galactosemic children compared with white children (5 ± 6 vs 1 ± 2 μmol/hr per gram, respectively) (normal 172 to 374 μmol/hr per gram), but the difference was not statistically significant ( p = 0.11). Analysis by genotype revealed that the two S135L homozygotes had much more leukocyte activity (9 and 17 μmol/hr per gram) than Q188R homozygotes or than all non-S135L allelic genotypes. Compound heterozygotes (S135L/G) had intermediate activity. The GALT protein was not detectable by Western blot in the erythrocytes of either white or black galactosemic children, as determined by antibodies specific for both C- and N-terminal sequences. The GALT protein was undetectable in the leukocytes of white galactosemic children, but leukocytes from black galactosemic children with the S135L mutation contained reduced but readily detectable GALT protein. Erythrocyte galactose 1-phosphate levels were significantly lower in galactosemic children with an S135L mutant allele (1.1 ± 0.2 gm/dl) compared with children who had other mutations (3.1 ± 0.9 mg/dl; p = 0.0001). The correlation of protein content data with activity levels in the blood cells suggests that the S135L missense mutation affects the stability of GALT protein to produce a deficiency state. (J Pediatr 1997;130:972-80)

13-cis-Retinoic acid (13-CRA), a water-soluble vitamin A analog and 5'-lipoxygenase inhibitor, was tested in vitro for effects on excess oxidative metabolism and DNA damage in mitogen-stimulated lymphocytes from patients with systemic lupus erythematosus (SLE), because other 5'-lipoxygenase enzyme inhibitors were shown to lower the excess oxidative metabolism in SLE cells. Excess chemiluminescence (CL) was abolished within minutes after the addition of 1 x 10(-6) M 13-CRA in five of five CL-positive mitogen-stimulated SLE lymphocytes, and was lowered in five of eight samples after 48 to 72 h culture. Similarly, low concentrations of 13-CRA for 48-72 h largely prevented the S1 nuclease-sensitive DNA changes/DNA damage observed in CL-positive lupus lymphocytes in vitro. However, 13-CRA did not affect DNA damage in four of four CL-negative lymphocyte samples. 13-CRA, like other retinoic acid compounds, was known to stimulate B-cell activities in vivo and in vitro but effects on dividing lupus T cells had not been studied. 13-CRA further inhibited the diminished PHA-stimulated lupus T-cell growth in tissue culture at a concentration of 9 x 10(-6) M in three of five lupus lymphocyte samples. 13-CRA has positive and negative effects on multiple aspects of the immune system and it is not clear whether 13-CRA will have positive or adverse clinical effects on SLE patients. Close attention to vitamin A and vitamin "supplements" in patients with SLE may answer this question.

textabstractBackground: Fragile X syndrome (FXS) is caused by loss of function mutations in the FMR1 gene. Trinucleotide CGG-repeat expansions, resulting in FMR1 gene silencing, are the most common mutations observed at this locus. Even though the repeat expansion mutation is a functional null mutation, few conventional mutations have been identified at this locus, largely due to the clinical laboratory focus on the repeat tract. Methodology/Principal Findings: To more thoroughly evaluate the frequency of conventional mutations in FXS-like patients, we used an array-based method to sequence FMR1 in 51 unrelated males exhibiting several features characteristic of FXS but with normal CGG-repeat tracts of FMR1. One patient was identified with a deletion in FMR1, but none of the patients were found to have other conventional mutations. Conclusions/Significance: These data suggest that missense mutations in FMR1 are not a common cause of the FXS phenotype in patients who have normal-length CGG-repeat tracts. However, screening for small deletions of FMR1 may be of clinically utility.

Gain-of-function mutations of NaV1.7 have been shown to produce two distinct disorders: NaV1.7 mutations which enhance activation produce inherited erythromelalgia (IEM), characterized by burning pain in the extremities; NaV1.7 mutations which impair inactivation produce a different, non-overlapping syndrome, paroxysmal extreme pain disorder (PEPD), characterized by rectal, periocular and perimandibular pain. Here we report a novel NaV1.7 mutation associated with a mixed clinical phenotype with characteristics of IEM and PEPD, with an alanine 1632 substitution by glutamate (A1632E) in domain IV S4-S5 linker. Patch-clamp analysis shows that A1632E produces changes in channel function seen in both IEM and PEPD mutations: A1632E hyperpolarizes (-7 mV) the voltage-dependence of activation, slows deactivation, and enhances ramp responses, as observed in NaV1.7 mutations that produce IEM. A1632E depolarizes (+17mV) the voltage-dependence of fast-inactivation, slows fast-inactivation and prevents full inactivation, resulting in persistent inward currents similar to PEPD mutations. Using current-clamp, we show that A1632E renders dorsal root ganglion (DRG) and trigeminal ganglion neurons hyperexcitable. These results demonstrate a NaV1.7 mutant with biophysical characteristics common to PEPD (impaired fast-inactivation) and IEM (hyperpolarized activation, slow deactivation and enhanced ramp currents) associated with a clinical phenotype with characteristics of both IEM and PEPD, and show that this mutation renders DRG and trigeminal ganglion neurons hyperexcitable. These observations indicate that IEM and PEPD mutants are part of a physiological continuum that can produce a continuum of clinical phenotypes.

We have examined oxidative metabolism in phytohemagglutinin (PHA)-stimulated lymphocytes from patients with systemic lupus erythematosus (SLE) because increased oxygen free radicals would explain the DNA abnormality previously observed in these cells. Almost no oxidative activity was found in freshly isolated control or lupus lymphocytes or control lymphocytes stimulated with PHA. However, increased oxidative metabolism, measured by nitroblue tetrazolium (NBT) conversion to formazan, was found in PHA-stimulated lymphocytes from 14 of 21 lupus patients. A time course study showed that NBT activity appeared in positive lupus lymphocytes at 1–2 days of PHA stimulation, increased to a maximum at 2–4 days, and diminished thereafter. NBT activity was not related to specific disease symptoms, drug therapy, or serum dsDNA, Sm, RNP, or SSB (La) antibodies. The selected population of lupus patients studied precluded conclusions about NBT activity and disease severity. However, the intensity of NBT response in stimulated lupus lymphocytes was positively correlated with the presence of serum SSA (Ro) antibody. We suggest that increased oxidative activity of SLE lymphocytes generates a chemical change in endogenous DNA in vivo and may be a primary event in the pathogenesis of autoimmunity. Absence of detectable oxidative activity in stimulated lymphocytes in a subgroup of lupus patients suggests that at least two different mechanisms are associated with the altered DNA profiles observed in this disorder.

Factors that potentially affect the generation of excess low molecular weight DNA (LMW-DNA) in cultured phytohemagglutinin (PHA)-stimulated lymphocytes of patients with systemic lupus erythematosus (SLE) were studied because this species of DNA is consistently found and this DNA may play a role in the pathogenesis of the disease. Superoxide dismutase (SOD; 0.05 mg/mL), a scavenger of free radical oxygen, decrease LMW-DNA formation in lymphocytes by 22%. Co-cultivation with cysteamine, a second scavenger of free radical oxygen and a sulfhydryl radioprotective agent, resulted in a 32% decrease in the generation of excess LMW-DNA at a concentration of 0.5 x 10(-3) mol/L and largely prevented its formation at 1.0 x 10(-3) mol/L. Other free radical scavengers (catalase, mannitol, vitamins C and E), cyclooxygenase inhibitors (ibuprofen and aspirin), a xanthine oxidase inhibitor (allopurinol), and an iron chelator (desferoxamine) did not affect excess LMW-DNA formation. Glutathione (1 x 10(-3) mol/L) had no effect and cysteine was toxic. Because scavengers of free radicals might be useful in the therapy of lupus, a trial of cysteamine (30 to 60 mg/kg/d) was administered to six acutely ill patients with SLE. A therapeutic benefit was not demonstrated, and some patients had exacerbation of disease. Lymphocyte cell growth from control and lupus subjects was stimulated when cysteamine, 1 x 10(-5) to 1 x 10(-4) mol/L was added to the media, but inhibited at concentrations of 2 x 10(-4) mol/L or greater. These studies suggest that the autooxidation and toxicity of high-dose cysteamine preclude its therapeutic use as a free radical scavenger.

Genomic imbalance is a major cause of developmental disorders. Microarray-based comparative genomic hybridization (aCGH) has revealed frequent imbalances associated with clinical syndromes, but also a large number of copy number variations (CNVs), which have complicated the interpretation of results. We studied 100 consecutive patients with unexplained mental retardation and a normal karyotype using several platforms of CGH arrays. A genomewide array with 44,290 oligonucleotide probes (OaCGH44K) detected imbalances in 15% of cases studied with sizes ranged from 459 kb to 19 Mb while revealing a small number of CNVs (0.72/individual). Another platform with approximately 240,000 oligonucleotide probes (OaCGH244K) revealed a large number of CNVs (20/individual) in selected cases and their normal parents. We used a comprehensive approach for interpreting the results of aCGH, including consideration of the size, inheritance and gene content of CNVs, and consultation with an online Database of Genomic Variants (DGV) and Online Mendelian Inheritance in Men (OMIM) for information on the genes involved. Our study suggests that genomewide oligonucleotide arrays such as the OaCGH44K platform can be used as a powerful diagnostic tool for detection of genomic imbalances associated with unexplained mental retardation or syndromic autism spectrum disorders. It is interesting to note that a small number of common variants were revealed by OaCGH244K in some study subjects but not in their parents and that some inherited CNVs had altered breakpoints. Further investigations on these alterations may provide useful information for understanding the mechanism of CNVs.

We present a case of 46,XX sex reversal in the absence of SRY but with partial duplication of chromosome 22q. The subject had multiple congenital anomalies but nearly complete masculinization of the external genitalia. Our case along with a previous case supports the existence of a gene on chromosome 22q that can trigger testis determination in the absence of SRY. We proposed that overexpression of the SOX10 gene at 22q13 might be the cause of sex reversal. We investigated 13 additional subjects with SRY-negative 46,XX sex reversal for microduplication of chromosome arm 22q in the region of SOX10 gene, but could not find evidence for it.

X-linked ocular albinism (OA1), Nettleship-Falls type, is characterized by decreased ocular pigmentation, foveal hypoplasia, nystagmus, photodysphoria, and reduced visual acuity. Affected males usually demonstrate melanin macroglobules on skin biopsy. We now report results of deletion and mutation screening of the full-length OA1 gene in 29 unrelated North American and Australian X-linked ocular albinism (OA) probands, including five with additional, nonocular phenotypic abnormalities (Schnur et al. 1994). We detected 13 intragenic gene deletions, including 3 of exon 1, 2 of exon 2, 2 of exon 4, and 6 others, which span exons 2-8. Eight new missense mutations were identified, which cluster within exons 1, 2, 3, and 6 in conserved and/or putative transmembrane domains of the protein. There was also a splice acceptor-site mutation, a nonsense mutation, a single base deletion, and a previously reported 17-bp exon 1 deletion. All patients with nonocular phenotypic abnormalities had detectable mutations. In summary, 26 (approximately 90%) of 29 probands had detectable alterations of OA1, thus confirming that OA1 is the major locus for X-linked OA.

A non-progressive recessive cerebellar ataxia was identified in a highly inbred Cayman island population. Cayman cerebellar ataxia is characterized by marked psychomotor retardation, and prominent cerebellar dysfunction manifested by nystagmus, intention tremor, dysarthric speech, and an ataxic gait. In this study, we identify linkage to chromosome 19p 13.3 using pooled DNA samples of affected individuals from an isolated population as PCR template for a genome wide screen with short tandem repeat markers. Our data demonstrate that the DNA pooling approach to identify disease gene loci is feasible using individuals from isolated populations in which kindred relationships are highly complex and exact relationships between all affected individuals are not known. Genetic fine mapping demonstrates that the genetic disease interval is approximately 9 cM, but contained within a small physical region. The existence of multiple individuals that are recombinant with flanking markers indicates that the disease interval can be further narrowed with additional markers.

Chemiluminescence (CL) was examined in phytohemaglutinin (PHA)-stimulated control and lupus lymphocytes because oxidative radicals have the chemical potential to generate DNA changes recently observed in lupus lymphocytes. Increased CL was found in 30 of 65 PHA-stimulated lupus lymphocyte samples by a luminol assay. CL did not correspond statistically to oxidative potential measured by a nitroblue tetrazolium assay. CL did not appear to be related to disease activity, organ involvement, or drug therapy. However, six of six males tested had positive CL activity. Cocultivation of CL-positive PHA-stimulated lupus lymphocytes with metabolic inhibitors of various oxidative enzymes revealed that 50 μ m arachidonic acid dramatically inhibited the excess oxidation. A specific inhibitor of 5-lipoxygenase activity, 3 μ m nordihydroguaiaretic acid, abolished excess CL activity. These studies suggest that chemiluminescence assays can be used to better understand the oxidative metabolism in lupus lymphocytes. The enzyme 5-lipoxygenase may be dysfunctional in a subgroup of lupus patients.

DNA samples from control and lupus lymphocytes were studied for DNA integrity and single-strand breaks by agarose gel electrophoresis following digestion with the enzyme S1 nuclease. S1 nuclease digests single-strand gaps in double-stranded DNA. Gel patterns of phytohemagglutinin (PHA)-stimulated control and lupus lymphocyte DNAs were identical in the absence of S1 nuclease incubation. DNA isolated from PHA-stimulated control lymphocytes was relatively resistant to S1 nuclease digestion in 14 of 16 samples. However, 15 of 16 DNA samples from PHA-stimulated lupus lymphocytes demonstrated dramatically greater S1 nuclease digestion than paired control DNAs from lymphocytes analyzed at the same time under the same conditions. Increased S1 sensitivity suggests that more single-strand DNA breaks were found in PHA-stimulated lupus lymphocytes and/or the lupus DNA was more damaged than control DNA. We suggest that structural changes found in DNA from stimulated T lymphocytes of lupus patients are consistent with an endogenous antigen-mediated disorder.

All patients with systemic lupus erythematosus (SLE) demonstrated two classes of newly synthesized DNA in sucrose density gradients of PHA (phytohemagglutinin)-stimulated lymphocytes: a large-molecular-weight fraction that comigrates with control DNA and an excess low-molecularweight DNA (LMW-DNA) fraction not found in control lymphocytes. Excess LMW-DNA was independent of disease activity or drug therapy. LMW-DNA and serologic abnormalities were studied in a four-generation family in which two first cousins had SLE. Excess LMW-DNA was found in the cousins with SLE, sibling parents of the SLE patients, a common grandparent, four of nine siblings of one patient, and five of seven at risk children. Both males and females had excess LMW-DNA. Male-male transmission was observed. The expression of excess LMW-DNA in stimulated lymphocytes is inherited as an autosomal dominant genetic trait in this family. All unaffected adult family members with the marker had positive antinuclear antibodies (ANAs) except the grandmother. However, none of the five children with excess LMW-DNA showed positive ANAs. Excess LMW-DNA precedes the appearance of ANAs when found in children of adults with excess LMW-DNA, and may be a predisposing factor in the development of the immunologic responses of systemic lupus erythematosus.

Nat. Genet. 35, 264–269 (2003). This paper states that “affected subjects and family members...gave informed consent approved by the University of Miami Institutional Review Board.” This is an error. Informed consent was approved by the Minister of Health, Grand Cayman Islands, and the Research Committee of the Grand Cayman Islands Government, and all families and all subjects able to do so gave informed consent.

Early studies suggested that African American children (AA) with galactosemia developed fewer symptoms than white galactosemic children (W). The biochemical and molecular basis for this disparity has not been clarified. Galactosemia is caused by deficiency of galactose-1-phosphate uridyl transferase (GALT). In this study, GALT activity was measured in the RBC and WBC of 10 W and 8 AA. WBC activities were measured because WBC unlike RBC make GALT protein and may therefore more accurately reflect GALT activity in other tissues. GALT protein content was determined by Western blot. Mutational analysis of the gene was undertaken to determine the presence of Q188R (common mutation) and S135L (found only in AA) alleles. S135L was present in 9/16 AA alleles.

Male and female siblings demonstrated similar facial features and had seizures from birth. Neurologic development, which was delayed, began to deteriorate at 1 year. Sudden death occurred at 2 8/12 and 2 3/12 years of age associated with respiratory infections. Tanning of the skin was noted 2 months before death in the first child. In the second child, blood cortisol levels failed to increase after intravenous ACTH administration, and computerized axial tomography (CAT) scans were normal. At autopsy both patients demonstrated adrenal atrophy and degenerative changes of the white matter throughout the neuraxis. We propose that these siblings have a new form of adrenoleukodystrophy that can be distinguished from the X-linked form by onset at birth, clinical appearance, and pattern of inheritance. A comparison of these cases with a second disorder, Zellweger's syndrome, suggests that a distinctive phenotype is associated with intrauterine degeneration of white matter.