Gerard T. Berry

Deletions of NRXN1 (Neurexin-1) Predispose to a Wide Spectrum of Developmental Disorders

Research has implicated mutations in the gene for neurexin‐1 (NRXN1) in a variety of conditions including autism, schizophrenia, and nicotine dependence. To our knowledge, there have been no published reports describing the breadth of the phenotype associated with mutations in NRXN1. We present a medical record review of subjects with deletions involving exonic sequences of NRXN1. We ascertained cases from 3,540 individuals referred clinically for comparative genomic hybridization testing from March 2007 to January 2009. Twelve subjects were identified with exonic deletions. The phenotype of individuals with NRXN1 deletion is variable and includes autism spectrum disorders, mental retardation, language delays, and hypotonia. There was a statistically significant increase in NRXN1 deletion in our clinical sample compared to control populations described in the literature (P = 8.9 × 10−7). Three additional subjects with NRXN1 deletions and autism were identified through the Homozygosity Mapping Collaborative for Autism, and this deletion segregated with the phenotype. Our study indicates that deletions of NRXN1 predispose to a wide spectrum of developmental disorders.

Transcription of the Sodium/myo-Inositol Cotransporter Gene Is Regulated by Multiple Tonicity-responsive Enhancers Spread over 50 Kilobase Pairs in the 5'-Flanking Region *

The sodium/myo-inositol cotransporter is a plasma membrane protein responsible for concentrative cellular accumulation of myo-inositol in a variety of tissues. When cells in kidney and brain are exposed to a hyperosmolar salt condition (hypertonicity) due to the operation of urinary concentration mechanism and pathological conditions, respectively, they survive the stress of hypertonicity by raising the cellular concentration ofmyo-inositol. Transcription of the sodium/myo-inositol cotransporter gene is markedly stimulated in response to hypertonicity, leading to an increase in the activity of the cotransporter, which in turn drives the osmoprotective accumulation of myo-inositol. To understand the molecular mechanisms by which hypertonicity stimulates transcription, we analyzed the 5′-flanking region of the cotransporter gene forcis-acting regulatory sequences. We identified five tonicity-responsive enhancers that are scattered over 50 kilobase pairs. All the enhancers are variations of the same type of enhancer interacting with the transcription factor named tonicity-responsive enhancer binding protein. In vivo methylation experiments demonstrated that exposure of cells to hypertonicity increases the binding of tonicity-responsive enhancer binding protein to the enhancer sites, indicating that all of these enhancers are involved in the transcriptional stimulation. We conclude that the sodium/myo-inositol cotransporter gene is regulated by a large region (∼50 kilobase pairs) upstream of the gene.

Exploring concordance and discordance for return of incidental findings from clinical sequencing

Purpose:The aim of this study was to explore specific conditions and types of genetic variants that specialists in genetics recommend should be returned as incidental findings in clinical sequencing.Methods:Sixteen specialists in clinical genetics and/or molecular medicine selected variants in 99 common conditions to return to the ordering physician if discovered incidentally through whole-genome sequencing. For most conditions, the specialists independently considered three molecular scenarios for both adults and minor children: a known pathogenic mutation, a truncating variant presumed pathogenic (where other truncating variants are known to be pathogenic), and a missense variant predicted in silico to be pathogenic.Results:On average, for adults and children, respectively, each specialist selected 83.5 and 79.0 conditions or genes of 99 in the known pathogenic mutation categories, 57.0 and 53.5 of 72 in the truncating variant categories, and 33.4 and 29.7 of 72 in the missense variant categories. Concordance in favor of disclosure within the adult/known pathogenic mutation category was 100% for 21 conditions or genes and 80% or higher for 64 conditions or genes.Conclusion:Specialists were highly concordant for the return of findings for 64 conditions or genes if discovered incidentally during whole-exome sequencing or whole-genome sequencing.Genet Med 2012:14(4):405–410

Clinical spectrum of succinic semialdehyde dehydrogenase deficiency.

Succinic semialdehyde dehydrogenase (SSADH) deficiency is a rare autosomal recessive disorder affecting CNS γ-aminobutyric acid (GABA) degradation. SSADH, in conjunction with GABA transaminase, converts GABA to succinate. In the absence of SSADH, GABA is converted to 4-OH-butyrate. The presence of 4-OH-butyrate, a highly volatile compound, may be undetected on routine organic acid analysis. Urine organic acid testing was modified at the authors’ institution in 1999 to screen for the excretion of 4-OH-butyrate by selective ion monitoring gas chromatography-mass spectrometry in addition to total ion chromatography. Since then, five patients with 4-hydroxybutyric aciduria have been identified. The authors add the clinical, neuroimaging, and EEG findings from a new cohort of patients to 51 patients reported in the literature with clinical details. Ages ranged from 1 to 21 years at diagnosis. Clinical findings include mild-moderate mental retardation, disproportionate language dysfunction, hypotonia, hyporeflexia, autistic behaviors, seizures, and hallucinations. Brain MRI performed in five patients at the authors’ institution revealed symmetric increased T2 signal in the globus pallidi. SSADH deficiency is an under-recognized, potentially manageable neurometabolic disorder. Urine organic acid analysis should include a sensitive method for the detection of 4-hydroxybutyrate and should be obtained from patients with mental retardation or neuropsychiatric disturbance of unknown etiology.

Endogenous synthesis of galactose in normal men and patients with hereditary galactosaemia

Despite restricted ingestion of lactose, patients with galactose-1-phosphate uridyltransferase deficiency have raised concentrations of galactose metabolites in blood and urine. Endogenous production of galactose may underlie this phenomenon. Using isotopically labelled galactose in a continuous intravenous infusion, we employed the steady-state flux method to calculate endogenous galactose production rate in three normal men and three patients with classic galactosaemia. We found that galactosaemic patients and normal subjects synthesise gram quantities of galactose per day. The rate of synthesis ranged from 0.53-1.05 mg/kg per h. Endogenous production of galactose may be an important factor in the pathogenesis of the complications of the brain and ovary, and could explain the persistent elevation of galactose metabolites in patients despite dietary restriction of galactose.

A new mutation associated with MELAS is located in a mitochondrial DNA polypeptide-coding gene

We report a patient with mitochondrial encephalomyopathy, lactic acidosis and stroke-like episodes (MELAS) who harbored a novel missense mutation at mtDNA position 9957 in the gene specifying subunit III of cytochrome c oxidase (COX III). This T-->C transition converted Phe-251, a highly conserved amino acid in the C-terminus of the polypeptide, to Leu. The mutation, which was not present in 107 normal controls or in 57 patients with various mitochondrial diseases, was heteroplasmic in both muscle and blood of the proband and in blood from his asymptomatic mother. These results provide evidence that the MELAS clinical phenotype can be due not only to mutations in mtDNA-encoded tRNA genes, but in polypeptide-coding genes as well.

Elevated Methylmalonic Acid and Total Homocysteine Levels Show High Prevalence of Vitamin B12 Deficiency after Gastric Surgery

Elderly persons [1, 2] and persons who have had gastric surgery [3-11] are at increased risk for developing vitamin B12 (cobalamin) deficiency. The hematologic and neurologic manifestations of vitamin B12 deficiency have been well described; however, this deficiency often remains undetected, and some patients receive a misdiagnosis of Alzheimer disease, spinal cord compression, amyotrophic lateral sclerosis, or diabetic or alcoholic peripheral neuropathy [12]. Although megaloblastic anemia is usually reversible with vitamin B12 treatment, the neurologic injuries are reversible only if they are treated soon after their onset [12, 13]. In addition, as do patients with folate deficiency [14], patients with untreated vitamin B12 deficiency have elevated total homocysteine levels. Substantial biochemical and epidemiologic evidence now suggests that an elevated serum total homocysteine level contributes to the development of carotid artery stenosis, coronary artery disease, and peripheral vascular disease [15-18]. Thus, in theory at least, patients with untreated vitamin B12 deficiency may be at increased risk for developing atherosclerotic vascular disease. In the future, the prevalence of vitamin B12 deficiency in the aging population may be expected to increase. Among persons at major risk are those who had subtotal gastrectomy for ulcer disease between the 1930s [19] and 1974 [5, 7] (in 1974, the first histamine-2 blocker, cimetidine, was released [20]). It is not possible to determine how many Americans had gastric surgery during this period, but representative data from the University of Minnesota Hospital suggest that the number is large. At that hospital alone, 1550 patients had subtotal gastrectomy between 1938 and 1950 [4]. Throughout the United States, therefore, hundreds of thousands of patients probably had this surgery. A new operation, gastric bypass for obesity, is currently creating another cohort at risk for developing vitamin B12 deficiency [11]. As these cohorts age, an unknown number of persons will develop vitamin B12 deficiency, and clinicians caring for such persons currently have no accurate guidelines on which to base screening decisions. Previous prevalence estimates are unreliable because clinical manifestations are insensitive and radiodilution vitamin B12 assays were nonspecific [21, 22]. The Schilling test is unreliable after gastrectomy [8, 9], anemia is often absent in vitamin B12-deficient patients [2, 12, 23], and macrocytosis may be masked by coexisting iron deficiency [7, 24]. See editorial comment on pp 509-511. Recently, however, measurements of the metabolites from two vitamin B12-dependent pathways (Figure 1)serum methylmalonic acid [25] and total homocysteine [14]were shown to be highly sensitive detectors of vitamin B12 deficiency [26]. Two enzymes have a known requirement for vitamin B12: L-methylmalonyl-CoA mutase and methionine synthase [22]. Methionine synthase requires folate in addition to vitamin B12 for normal functioning. If the conversion of L-methylmalonyl-CoA to succinyl-CoA is impaired by a deficiency of the vitamin B12 cofactor adenosylcobalamin, the excess methylmalonyl-CoA is cleaved to methylmalonic acid and methylmalonic acid levels in the serum and urine are elevated [25]. Similarly, if the methylation of homocysteine to methionine is impaired by a deficiency of methylcobalamin or methyltetrahydrofolate, serum total homocysteine levels are elevated [14]. The metabolic pathways in which these two enzymes function are not always equally affected by vitamin B12 deficiency. At the time vitamin B12 deficiency is diagnosed, therefore, levels of methylmalonic acid, total homocysteine, or both may be elevated [22]. Figure 1. The two vitamin B12-dependent enzymes, L-methylmalonyl-CoA mutase (left) and methionine synthase (right). In vitamin B12-deficient patients, elevated levels of both serum methylmalonic acid and total homocysteine decrease promptly with adequate vitamin B12 therapy [22, 26]. However, in folate-deficient patients, total homocysteine levels return to normal only after folate replacement [22]. Therefore, in addition to serum vitamin B12 levels, we used methylmalonic acid, total homocysteine, and folate levels to determine whether the prevalence of vitamin B12 deficiency differed between persons who had had gastric surgery and those who had not. Methods Between September 1991 and March 1993, 65 patients who had had gastric surgery were identified at the Philadelphia Veterans Affairs Medical Center. These patients were identified either by review of gastrointestinal radiographs, surveys of the house-staff assigned to the medicine and surgery inpatient services, or referral of outpatients from physicians in the medical clinic. Four of the 65 patients were excluded: Three were receiving vitamin B12 therapy, and one had a hepatoma. Hepatoma can produce increased levels of vitamin B12-binding protein, which may complicate interpretation of serum vitamin B12 levels. Patients who had not had gastric surgery (controls) were drawn from 127 consecutive patients attending one authors Philadelphia Veterans Affairs Medical Center clinic between November 1992 and March 1993. One hundred seven controls participated, and 20 either declined to participate or did not complete the required blood tests. We determined the type of gastric surgery that had been done either from patient reporting or by reviewing radiologic, endoscopic, or surgical records. In most patients (51 of 61), we determined the year surgery had been done from patient report or chart review. For patients who could not provide the year of surgery but could specify the decade, we used the mid-decade year. For example, if the patient said that the surgery had been done in the 1950s, we recorded the year as 1955. Serum vitamin B12 and folate levels were determined at the Philadelphia Veterans Affairs Medical Center using a commercially available radioligand kit (Bio-Rad, Diagnostics Group, Hercules, California). In the hospitals laboratory, normal values for vitamin B12 and folate levels were 171 to 840 pmol/L and 5 to 39 nmol/L, respectively. The remaining serum samples were frozen at 20 C and were shipped to Denver so that serum methylmalonic acid and total homocysteine levels could be analyzed by the stable isotope dilution gas chromatography-mass spectrometry method [27-30]. The normal range for serum methylmalonic acid levels (determined in 50 normal blood donors 18 to 65 years of age) is 73 to 271 nmol/L, and the normal range for serum total homocysteine levels is 5.4 to 16.2 mol/L [22]. Vitamin B12 deficiency was defined as one of the following: 1) a serum vitamin B12 level less than 221 pmol/L and an elevated methylmalonic acid level; 2) a serum vitamin B12 level less than 221 pmol/L and a total homocysteine level that decreased after vitamin B12 therapy; or 3) in patients unavailable for treatment, a serum vitamin B12 level less than 221 pmol/L, a folate level greater than 9 nmol/L, and an elevated total homocysteine level. Hemoglobin level, hematocrit, and mean corpuscular volume were measured by automatic devices. Macrocytosis was defined as a mean corpuscular volume of 95 fL or less. The peripheral smears of 71% of patients (43 of 61) and 88% of controls (94 of 107) were reviewed by one hematologist who was blinded to each participants vitamin B12 level, hemoglobin level, hematocrit, and gastric surgery status. Hypersegmentation was defined as five neutrophils with five or more lobes or one neutrophil with six lobes per 100 cells counted. Treatment Vitamin B12 treatment generally consisted of daily intramuscular injections of 1000 g of vitamin B12 for 5 days, followed by monthly injections. Folic acid was given orally, 1 mg/d. Serum vitamin B12, folate, methylmalonic acid, and total homocysteine levels were measured 1 to 6 weeks after treatment. Statistical Analysis Data were examined to determine whether the variables were suitable for parametric analyses. Although relatively modest, the skew for the numeric variables necessitated that several variables be transformed to logs for entry into two-way analysis of variance or be subjected to nonparametric analyses. The comparison between patients and controls for levels of vitamin B12, folate, methylmalonic acid, and total homocysteine was done by two-factor analysis of variance on log-transformed variables. In each analysis of variance, race was included as a factor (along with study group) to control for possible race-by-group interactions. We used unpaired t-tests or Mann-Whitney U tests to do comparisons of other numeric variables, such as hemoglobin and mean corpuscular volume; comparisons between other groups, such as patients with a positive and patients with a negative peripheral blood smear; and comparisons between deficient and nondeficient patients. We used chi-square tests to compare groups on dichotomous variables (such as white patients compared with black patients). Spearman correlations were used to assess the association between the time since surgery and other variables. The Human Studies Subcommittee and the Research and Development Committee of the Philadelphia Veterans Affairs Medical Center approved the study. Results Clinical Characteristics The 61 patients (who had had gastric surgery) and 107 controls (who had not) were similar in the ratio of men to women (60:1 compared with 104:3), age, and race (Table 1). The indications for surgery included peptic ulcer disease (56 patients), obesity (3 patients), gastric cancer (3 patients [2 of whom had previously had surgery for peptic ulcer disease]), and gastric lymphoma (1 patient). The type of gastric surgery could be determined in 36 of 61 patients (59%). The types of surgery were Billroth II (23 patients), repair of perforated ulcer (6 patients), vagotomy and pyloroplasty (2 patients), gastric bypass or gastric banding for obesity (3 patients), Billroth I (1 patient),

The human osmoregulatory Na+/myo-inositol cotransporter gene (SLC5A3): molecular cloning and localization to chromosome 21

A human Na+/myo-inositol cotransporter (SLC5A3) gene was cloned; sequencing revealed a single intron-free open reading frame of 2157 nucleotides. Containing 718 amino acid residues, the predicted protein is highly homologous to the product of the canine osmoregulatory SLC5A3 gene. The SLC5A3 protein is number 3 of the solute carrier family 5 and was previously designated SMIT. Using fluorescence in situ hybridization, the human SLC5A3 gene was localized to band q22 on chromosome 21. Many tissues including brain demonstrate gene expression. The inability of a trisomic 21 cell to downregulate expression of three copies of this osmoregulatory gene could result in increased flux of both myo-inositol and Na+ across the plasma membrane. The potential consequences include perturbations in the cell membrane potential and tissue osmolyte levels. The SLC5A3 gene may play a role in the pathogenesis of Down syndrome.

The mutation spectrum of the facilitative glucose transporter gene SLC2A2 (GLUT2) in patients with Fanconi-Bickel syndrome

Abstract. We report a total of 23 novel mutations of the SLC2A2 (GLUT2) gene in 49 patients with a clinical diagnosis of Fanconi-Bickel syndrome (FBS). Molecular genetic analysis has now been performed in more than 50% of the 109 FBS cases from 88 families that we have been able to locate world-wide since the original report in 1949. In these 49 patients, 33 different SLC2A2 mutations (9 missense, 7 nonsense, 10 frameshift, 7 splice-site) have been detected. Thus, our results confirm that mutations of SLC2A2 are the basic defect in patients with FBS. Mutations of SLC2A2 were detected in historical FBS patients in whom some of the characteristic clinical features (hepatorenal glycogen accumulation, glucose and galactose intolerance, fasting hypoglycemia, a characteristic tubular nephropathy) and the effect of therapy were described for the first time. Mutations were also found in patients with atypical clinical signs such as intestinal malabsorption, failure to thrive, the absence of hepatomegaly, or renal hyperfiltration. No single prevalent SLC2A2 mutation was responsible for a significant number of cases. In a high percentage (74%) of FBS patients, the mutation is homozygous, so we conclude that the prevalence of SLC2A2 mutations is relatively low in most populations. No mutational hot spots within SLC2A2 or even within homologous sequences among the genes for facilitative glucose transporters were detected.

Cytochrome c oxidase-associated Leigh syndrome: Phenotypic features and pathogenetic speculations ☆

Fourteen new cases of cytochrome oxidase (COX)-associated Leigh syndrome (LS) are combined with 20 reported cases to describe the clinical, laboratory, and radiological features of this devastating metabolic condition. Three clinical stages are identified. Most patients have normal neurological development during the first 8-12 months (stage I). Somatic complaints are common, including chronic diarrhea, recurrent vomiting, anorexia, and decelerating body and head growth. The second stage evolves during late infancy and early childhood when motor regression becomes evident. Eye signs, altered breathing patterns, pyramidal, extrapyramidal, and cerebellar signs emerge and sudden clinical deterioration occurs during intercurrent infectious or metabolic stress. The last stage may extend from 2 to 10 years and is manifested by extreme hypotonia, swallowing difficulties and undernutrition. Feeding assistance is necessary and seizures may occur. The CSF lactate concentration is consistently elevated and MRI abnormalities are seen in the subcortical structures. COX deficiency affects most tissues, but is not always generalized. For example, 3 patients with a cardiomyopathy had normal COX activity in cultured skin fibroblasts. Nearly normal amounts of cross-reacting material are present by ELISA and immunoblot analyses. Parental consanguinity has been found in several families, the hereditary pattern is recessive and males are affected more commonly (2:1). The biomolecular abnormality causing COX deficiency in LS is unknown, but the available evidence implicates a nuclear-encoded protein that affects the structure or the stability of the holoenzyme complex.