William K. Seltzer

DNA microextraction from dried blood spots on filter paper blotters: potential applications to newborn screening

SummaryMicroextraction of DNA from dried blood specimens would ease specimen transport to centralized laboratory facilities for recombinant DNA diagnosis in the same manner as use of dried blood spots allowed the broad application of screening tests to newborn populations. A method is described which reproducibly yields 0.5μg DNA from the dried equivalent of 50μl whole blood. Though DNA yields decreased with storage of dried specimens at room temperature, good-quality DNA was still obtained. Sufficient DNA was routinely obtained for Southern blot analysis using repetitive and unique sequences. This microextraction procedure will allow immediate application of molecular genetic technology to direct newborn screening follow-up of disorders amenable to DNA diagnosis, such as sickle cell anemia, and may eventually permit primary DNA screening for specific mutations.

A multicenter study of the frequency and distribution of GJB2 and GJB6 mutations in a large North American cohort

Purpose: The aim of the study was to determine the actual GJB2 and GJB6 mutation frequencies in North America after several years of generalized testing for autosomal recessive nonsyndromic sensorineural hearing loss to help guide diagnostic testing algorithms, especially in light of molecular diagnostic follow-up to universal newborn hearing screening.Methods: Mutation types, frequencies, ethnic distributions, and genotype-phenotype correlations for GJB2 and GJB6 were assessed in a very large North American cohort.Results: GJB2 variants were identified in 1796 (24.3%) of the 7401 individuals examined, with 399 (5.4%) homozygous and 429 (5.8%) compound heterozygous. GJB6 deletion testing was performed in 12.0% (888/7401) of all cases. The >300-kb deletion was identified in only nine individuals (1.0%), all of whom were compound heterozygous for mutations in GJB2 and GJB6. Among a total of 139 GJB2 variants identified, 53 (38.1%) were previously unreported, presumably representing novel pathogenic or benign variants.Conclusions: The frequency and distribution of sequence changes in GJB2 and GJB6 in North America differ from those previously reported, suggesting a considerable role for loci other than GJB2 and GJB6 in the etiology of autosomal recessive nonsyndromic sensorineural hearing loss, with minimal prevalence of the GJB6 deletion.Purpose: To determine short–term effects of intravitreal bevacizumab for subfoveal choroidal neovascularization (CNV) in pathologic myopia. Methods: In this prospective interventional case series, patients were treated with 2.5 mg of intravitreal bevacizumab and followed for 3 months. Best-corrected visual acuity (BCVA), optical coherence tomography (OCT), and fluorescein angiography (FA) were recorded. Indications for retreatment were active leaking CNV shown by FA and presence of subretinal fluid by OCT in combination with visual disturbances. Results: Fourteen patients were included, with a mean age of 53.86 ± 16.26 years (range 29–85). Mean spherical equivalent was −13.87 ± 3.68 diopters (−7.25 to −20.50). Minimum follow-up was 3 months. There were no adverse events. The mean initial visual acuity was 20/200 improving to 20/100 at 2 weeks, 20/80 at 4 weeks, and 20/60 at 8 and 12 weeks (P = 0.007; P = 0.001; P = 0.005; P = 0.001, respectively). Initial foveal thickness improved from 385.43 &mgr;m ± 125.83 &mgr;m to 257.64 ± 76.6 &mgr;m and 194.54 ± 54.35 &mgr;m after the first and third month, respectively (P = 0.001). Conclusions: Initial treatment results of patients with CNV due to pathologic myopia did not reveal any short-term safety concerns. Intravitreal bevacizumab resulted in a significant decrease in foveal thickness and improvement in visual acuity. These favorable initial results support further larger and long-term studies.

Laboratory Guidelines for Huntington Disease Genetic Testing

Martha A. Nance, Hennepin County Medical Center, Minneapolis (cochair); William Seltzer, Athena Diagnostics, Worcester, Massachusetts (cochair); Tetsuo Ashizawa, Baylor College of Medicine, Houston; Robin Bennett, University of Washington, Seattle; Nathalie McIntosh, DIANON Systems, Stratford, Connecticut; Richard H. Myers, Boston University School of Medicine, Boston; Nicholas T. Potter, University of Tennessee, Knoxville; and David K. Shea, Foundation for the Care and Cure of Huntington Disease, Islamorada, Florida.

An empirical estimate of carrier frequencies for 400+ causal Mendelian variants: results from an ethnically diverse clinical sample of 23,453 individuals

Purpose:Recent developments in genomics have led to expanded carrier screening panels capable of assessing hundreds of causal mutations for genetic disease. This new technology enables simultaneous measurement of carrier frequencies for many diseases. As the resultant rank-ordering of carrier frequencies impacts the design and prioritization of screening programs, the accuracy of this ranking is a public health concern.Methods:A total of 23,453 individuals from many obstetric, genetics, and infertility clinics were referred for routine recessive disease carrier screening. Multiplex carrier screening was performed and results were aggregated for this study.Results:Twenty-four percent of individuals were identified as carriers for at least one of 108 disorders, and 5.2% were carriers for multiple disorders. We report tabulations of carrier frequency by self-identified ethnicity and disease.Conclusion:To our knowledge, this study of a large, ethnically diverse clinical sample provides the most accurate measurements to date of carrier frequencies for hundreds of recessive alleles. The study also yields information on the clinical considerations associated with routine use of expanded panels and provides support for a pan-ethnic screening paradigm that minimizes the use of “racial” categories by the physician, as recommended by recent guidelines.Genet Med 2013:15(3):178–186

Huntington's disease–like 2 (HDL2) in North America and Japan

Huntingtons Disease–like 2 (HDL2) is a progressive, autosomal dominant, neurodegenerative disorder with marked clinical and pathological similarities to Huntingtons disease (HD). The causal mutation is a CTG/CAG expansion mutation on chromosome 16q24.3, in a variably spliced exon of junctophilin‐3. The frequency of HDL2 was determined in nine independent series of patients referred for HD testing or selected for the presence of an HD‐like phenotype in North America or Japan. The repeat length, ancestry, and age of onset of all North American HDL2 cases were determined. The results show that HDL2 is very rare, with a frequency of 0 to 15% among patients in the nine case series with an HD‐like presentation who do not have the HD mutation. HDL2 is predominantly, and perhaps exclusively, found in individuals of African ancestry. Repeat expansions ranged from 44 to 57 triplets, with length instability in maternal transmission detected in a repeat of 33 triplets. A younger age of onset is correlated with a longer repeat length (r2 = 0.29, p = 0.0098). The results further support the evidence that the repeat expansion at the chromosome 16q24.3 locus is the direct cause of HDL2 and provide preliminary guidelines for the genetic testing of patients with an HD‐like phenotype. Ann Neurol 2004

Screening for cystic fibrosis: Feasibility of molecular genetic analysis of dried blood specimens

Direct genotypic analysis for the common Caucasian cystic fibrosis mutation (delta F508) was performed using dried blood specimens in a filter paper matrix (neonatal screening blotter). DNA was obtained from dried and liquid blood samples, amplified, and analyzed by polyacrylamide gel electrophoresis. Additionally, intact 4-mm-diameter punched discs from blotters containing dried blood specimen were used in the amplification reactions and analyzed by electrophoresis. The results agreed completely between these three sample types, demonstrating the feasibility of molecular genetic confirmation of the delta F508 mutation from the neonatal screening blotter among those with positive CF screening results. Such a program could reduce follow-up testing by at least 50% in a CF newborn screening program and would identify immediately those families who would benefit from carrier detection for the delta F508 allele.

Characterization of patients with glycerol kinase deficiency utilizing cDNA probes for the Duchenne muscular dystrophy locus.

SummaryGenomic DNA from five previously unreported patients with glycerol kinase deficiency (GKD), dystrophic myopathy, and adrenal insufficiency were studied with genomic probes and cDNA probes for the Duchenne muscular dystrophy (DMD) locus. These individuals, together with those reported by ourselves and others, show that patients with a contigous gene syndrome involving the DMD, GK, and adrenal hypoplasia congenita (AHC) loci have a broader distribution of microdeletion breakpoints than those observed among patients with classical DMD. This study demonstrates the use of the DMD cDNA probes to delineate the centromeric deletion breakpoints for patients with Xp21 microdeletions extending beyond the DMD locus. It also shows the practical diagnostic application of the DMD cDNA probes when the diagnosis of GKD is entertained in a patient with known DMD and only DNA is available for study.

Subcellular distribution and kinetic properties of soluble and particulate-associated bovine adrenal 21vcerol kinase

SummaryThe subcellular distribution and substrate kinetics of soluble and particulate-associated bovine adrenal glycerol kinase have been investigated. Whole adrenal, adrenal cortex and adrenal medulla were examined for distribution of glycerol kinase between soluble and particulate fractions. No major differences in distribution were noted between these tissues; of the total homogenate activity, 0–20% sedimented with the nuclear fraction, 24–36% sedimented with the post-nuclear fraction and 62–69% remained soluble. Steadystate kinetic parameters of glycerol kinase activity were compared in the soluble and mitochondrial fractions. The Km for glycerol in the soluble fraction was 6.3 ± 0.1 μM and in the mitochondrial fraction was 4.0 = 0.3 μM. The Km for ATP in soluble fraction was 12.8 Λ 1.5 and in the mitochondrial fraction was 5.3 ± 1.6. Release of adrenal glycerol kinase from the mitochondria) fraction was investigated using inorganic phosphate, ATP and glycerol 3-phosphate. Of these compounds, only ATP and glycerol 3-phosphate were effective in releasing particulate-associated glycerol kinase. Inorganic phosphate had no effect upon release. Particulate-associated glycerol kinase activity of the mitochondrial fraction was stimulated by addition of succinate and ADP and was inhibited by addition of atractyloside. The data presented here indicate that bound glycerol kinase found within the mitochondrial fraction is kinetically distinct from soluble glycerol kinase and binding to mitochondria is responsive to substrate and product levels within the physiological range.

Immunoreactive trypsinogen levels in infants with cystic fibrosis complicated by meconium ileus

Abstract To examine abnormalities in immunoreactive trypsinogen (IRT) in infants with cystic fibrosis (CF) and meconium ileus (MI) and to evaluate the utility of IRT as a diagnostic aid in MI, we compared IRT in 19 infants with CF and MI to values in normal infants and in 91 infants with CF without MI. Infants with CF without MI were identified between 1982–1989 through a statewide screening program based on IRT measurements ( n =83) or through clinical presentation ( n = 8). Infants with MI were identified through clinical presentation during the same period. The IRT level of MI patients as a group (195±23 ng/ml) was greater than the cutoff value for IRT in normal infants (140 ng/ml, corresponding to the 99.7th percentile for infants less than 20 days of age; P P

Blood phenylalanine estimation for the patient with phenylketonuria using a portable device

A simple and reliable method is described which is suitable for estimation of a whole blood phenylalanine concentration for the patient with PKU in various settings including the physicians office and the home. Excellent correlations were obtained between this method and weighed phenylalanine standards, as well as with measurement of phenylalanine in serum, plasma, and whole blood, using the McCaman-Robins fluorometric assay. Increasing the frequency and rapidity of feedback to the patient should improve metabolic control, just as home glucose monitoring has for the patient with diabetes mellitus. This method is immediately adaptable to monitoring patients with tyrosinemia, and with substitution of the appropriate amino acid ammonia lyase could be used for other amino acidemias.