Karen Snow

Spinocerebellar ataxia type 2 (SCA 2) in an infant with extreme CAG repeat expansion

Autosomal dominant cerebellar ataxias are a heterogeneous group of neurodegenerative disorders that generally present in adulthood. Spinocerebellar ataxia type 2 typically presents with progressive cerebellar symptoms, slow ocular saccades, and peripheral neuropathy. The onset of symptoms is usually between 20 and 40 years. We describe an infant who presented with neonatal hypotonia, developmental delay, and dysphagia. Ocular findings of retinitis pigmentosa were noted at 10 months. Her father had mild spinocerebellar ataxia first noted at age 22 years. Molecular studies of the SCA2 gene showed a CAG expansion of 43 repeats in the father and an extreme CAG repeat expansion of more than 200 in the baby. Our report expands the known phenotype and genotype of SCA2. Testing for dominant ataxias should be included in the evaluation of infants with nonspecific progressive neurologic symptoms and retinitis pigmentosa, especially in cases with a positive family history for spinocerebellar ataxia.

Characteristics of two cases with dup(15) (q 11.2-q 12): one of maternal and one of paternal origin

Purpose: The phenotype correlations for interstitial duplications that include the Prader-Willi/Angelman syndrome critical region are not well established. We describe two such duplication cases, one of which was of maternal origin and the other was paternal.Methods: High resolution G-banding, fluorescence in situ hybridization (FISH) for SNRP-N and D15S10 were used for cytogenetic analysis. Southern blot analyses based on parent of origin specific DNA methylation at D15S63 (PW71) locus were utilized for detection of methylated and unmethylated fragments.Results: The duplication was established by the FISH analysis. The molecular pattern suggested a maternal origin of the duplication in patient 1 and a paternal origin in patient 2. Patient 1 (2 years old) had developmental and speech delays with pervasive developmental disorder or mild autism, strabismus, and normal growth parameters with seizures. Patient 2 (16 years old) had global developmental delay, verbal IQ of 94, depression, obesity, food-seeking behavior, and significant behavioral problems that included self-injurious tendencies. Neither patient had significant dysmorphic features or abnormalities of internal organs.Conclusion: The two cases suggest that some patients with 15g11.2g12 duplication may have significant anomalies, and there appear to be phenotypic differences between maternal and paternal transmission of the duplication.

Characterization of human sperm antigens and antisperm antibodies in infertile patients

Objective To identify which sperm antigens may elicit the production of functionally important antisperm antibodies. Design Immunoblot analysis was performed on 69 serum and 9 seminal plasma samples from infertile patients, using detergent extracts of pooled donor sperm as the antigen source. Serum and seminal plasma had been previously tested by an indirect immunobead binding test (IBT); 61 IBT-positive and 17 IBT-negative samples were included in the study. Proteins recognized by IBT-positive but not IBT-negative samples were most likely to be cell surface antigens, whereas proteins recognized by both IBT-positive and IBT-negative samples were probably intracellular. Antibodies directed toward surface antigens would be most likely to affect fertilization. Characterization of sperm surface proteins on both acrosome-intact and -reacted sperm used labeling of cell surface proteins with an N-hydroxysuccinimide ester of biotin, fractionation of sperm heads and tails, and lectin binding to determine glycosylation. Results Specific immunoreactivity (with respect to IBT results) was observed to 35K, 40 to 45K, 57K, 66K, and 88 to 90K MW proteins. Characterization studies identified an 88K MW glycosylated plasma membrane protein, a 66K MW inner acrosomal membrane protein, a 34K MW inner acrosomal membrane protein, and a 35K MW prominent tail protein. Conclusion Immunological infertility may involve several antigens characterized in this study. Further studies are necessary to determine if antibodies to these specific proteins interfere with sperm function.

Frontal lobe atrophy due to a mutation in the cholesterol binding protein HE1/NPC2

This is the first description of slowly progressive Niemann–Pick disease type C (NPC) without the typical lysosomal storage in bone marrow and viscera in two descendants of a group of 17th century French‐Canadians. The index patient was a married 43‐year‐old woman with onset of dementia in her thirties, later followed by the development of ataxia and athetoid movements. Her autopsy disclosed frontal lobe atrophy, neurolysosomal storage with oligolamellar inclusion and tau‐positive neurofibrillary tangles. Of the 119 family members screened, only a married 42‐year‐old sister displayed symptoms of a dementia. Both women displayed vertical supranuclear ophthalmoplegia; expressive aphasia; concrete, stimulus‐bound, perseverative behavior; and impaired conceptualization and planning. Cultured fibroblasts showed decreased cholesterol esterification and positive filipin staining, but no mutation was detected in coding or promoter regions of the NPC1 gene using conformation sensitive gel electrophoresis and sequencing. Sequencing showed a homozygous gene mutation that is predicted to result in an amino acid substitution, V39M, in the cholesterol binding protein HE1 (NPC2). Adult‐onset NPC2 with lysosomal storage virtually restricted to neurons represents a novel phenotypic and genotypic variant with diffuse cognitive impairment and focal frontal involvement described for the first time. Ann Neurol 2002;52:000–000

Molecular Diagnostics in Preimplantation Genetic Diagnosis

Preimplantation genetic diagnosis (PGD) is a procedure that allows embryos to be tested for genetic disorders before they enter the uterus and before pregnancy has begun. Embryos obtained by in vitro fertilization undergo a biopsy procedure in which one or two cells are removed and tested for a specific disorder. If the cell is unaffected, the embryo from which it was taken is judged to be free of the disorder. The embryo can then be transferred to the uterus to initiate pregnancy. Couples whose children are at increased risk for a specific genetic disorder can benefit from PGD. Some of these couples may have affected family members or family ancestry that puts them at high risk for transmitting a particular disorder to their offspring. PGD is an alternative to prenatal tests such as amniocentesis or chorionic villus sampling and since it is performed before a pregnancy has begun, it may be more acceptable to couples who have either had an affected child, previous termination of pregnancy, or who have objections to termination of pregnancy. PGD tests have largely focused on two methodologies: fluorescent in situ hybridization (FISH) and polymerase chain reaction (PCR). This review will focus on the use of PCR-based methodologies to diagnose single gene disorders in single cells; specifically describing the characteristics and limitations of single cell PCR and mutation detection strategies which have been developed for use in clinical PGD. The hundreds of cycles of preimplantation diagnosis performed to date have resulted in the birth of several hundred healthy children. 1 As shown in Table 1​1 , the genetic conditions for which PGD has been applied are numerous and the various methods used for diagnosis reflect the heterogeneity of causative mutations. Table 1. Strategies for PCR-Based Tests Used for Clinical Preimplantation Genetic Diagnosis The first clinical application of PGD used a generic PCR protocol for gender determination to avoid the transfer of male embryos which have a 50% probability of being affected by an X-linked recessive disorder. Gender was determined in a single blastomere by a single round of PCR using primers for Y-chromosome specific repetitive DNA sequences. The presence of Y-specific PCR amplification products was indicative of a male embryo and the absence of products was scored as female. 2 Although this approach had some success, a misdiagnosis, presumably due to amplification failure, did occur and emphasized the challenges inherent in single cell analysis and, more specifically, the danger in relying on the absence of amplification to diagnose genotype. 3 Subsequently, PCR protocols for preimplantation gender determination were refined to include primer sets which simultaneously amplify sequences common to both sex chromosomes (for example single copy genes such as ZFX/ZFY, 4 AMELX/AMELY, 5 ) and repetitive sequences such as DXZ1 and DYZ1. 6, 7 Sequences common to the sex chromosomes are identical at the site of primer annealing but differ internally in terms of size or include minor polymorphisms. Despite these technical improvements, the majority of embryo sexing is now accomplished using fluorescent in situ hybridization (FISH) which is less prone to contamination and can also provide the copy number for each chromosome tested thereby potentially avoiding the transfer of common chromosome abnormalities such as triploidy or X-monosomy. 8, 9 Although FISH has largely superseded PCR for sex determination, the specific diagnosis of single-gene defects remains dependent on DNA amplification with PCR. In the case of X-linked disorders, testing of the specific gene has the added advantage of ensuring that all embryos free of the mutant gene can be selected for transfer, irrespective of gender. 10, 11, 12 The list of disorders and the particular mutation detection strategies used for PCR-based clinical PGD application are given in Table 1​1 .

Mannose-binding lectin (MBL) deficiency. Variant alleles in a midwestern population of the United States.

OBJECTIVES To describe a method for the genotype analysis of mutations in the gene encoding mannose binding lectin (MBL), study the incidence of MBL gene mutations in a population of the Midwest of the United States, and compare it with previous reports in other populations. The objective of this report is also an extensive review of the literature to analyze the importance of MBL deficiency in human disease. DATA SOURCES Blood samples were obtained from the blood bank of the Mayo Clinic. They represented a population of blood donors living in the Midwest of the United States. A review of the literature was performed by selection of articles from Medline database. STUDY SELECTION Blood samples, 148, were randomly selected from a pool of blood donors. They included both females and males. Blood donors had been previously screened by a questionnaire and were found to be generally healthy. For the literature review, articles containing original data on MBL in humans were selected. RESULTS Forty-five (30.4%) of the analyzed blood donors carried one variant allele, while 8 donors (5.4%) showed homozygosity or compound heterozygosity for MBL gene mutations. Allele frequency for the different MBL variants is provided. Our results are similar to those reported for the Danish population. Literature review provides evidence for a significant role of MBL deficiency in the innate immunity. The incidence of MBL mutations is higher among patients with recurrent infections and autoimmune disorders. CONCLUSIONS Mannose binding lectin deficiency has a definite role in the pathogenesis of primary immunodeficiency in humans and screening patients with recurrent infections and autoimmunity might be beneficial. The significance of MBL deficiency among apparently healthy blood donors remains to be determined.

Hb DARTMOUTH [α66(E15)Leu → Pro (α2) (CTG → CCG)]: A NOVEL α2-GLOBIN GENE MUTATION ASSOCIATED WITH SEVERE NEONATAL ANEMIA WHEN INHERITED IN TRANS WITH SOUTHEAST ASIAN α-THALASSEMIA-1

We report a novel mutation at α66(E15)Leu → Pro (α2) (CTG → CCG), that we have named Hb Dartmouth for the medical center at which the patients were cared for, in monozygotic twins who also inherited the Southeast Asian α-thalassemia-1 deletion. The mother, of Khmer ancestry, is heterozygous for α-thalassemia-1. The father, who is of Scottish-Irish ancestry, is a silent carrier of the codon 66 mutation. The twins had severe neonatal anemia requiring transfusion.

Frequency and clinical significance of the S1235R mutation in the cystic fibrosis transmembrane conductance regulator gene: Results from a collaborative study

More than 900 mutations in the cystic fibrosis transmembrane conductance regulator (CFTR) gene have been reported to the cystic fibrosis (CF) consortium. A missense mutation, S1235R, was originally reported in a CF patient with a second mutation (G628R) on the same chromosome. The clinical significance of S1235R was not clear. S1235R is not among the commonly reported mutations, and it is not routinely screened for in most laboratories. However, we have detected the S1235R allele at a frequency that is significantly higher than that of many other CF mutations. Among more than 3,000 patients tested for either a possible diagnosis of CF or to determine CF carrier status, we identified 51 patients heterozygous for S1235R. No patients were homozygous for S1235R. Five patients were compound heterozygotes for a second CFTR mutation: two cases (one family) were N1303K/S1235R and three unrelated cases were ΔF508/S1235R. Our data suggest that S1235R, when combined with a second CF mutation, may be pathogenic, although phenotypic manifestations appear to be variable. The possibility that this represents a rare polymorphism cannot be discounted completely. Genetic counseling is difficult when S1235R is identified, even in the presence of a second known mutation, especially in prenatal cases. Am. J. Med. Genet. 95:361–365, 2000.

A search for germline APC mutations in early onset colorectal cancer or familial colorectal cancer with normal DNA mismatch repair

Twenty percent of colorectal cancers (CRCs) arise in people who have a family history of CRC in at least one other relative. Although a fraction of these CRCs are explained by two well‐described autosomal dominant syndromes—5% by hereditary nonpolyposis colorectal cancer (HNPCC) and 1% by familial adenomatous polyposis (FAP)—the cause of the remaining 14% of familial aggregates of CRC is unknown. Many cases of HNPCC are due to germline mutations in DNA mismatch repair genes, leading to the tumor phenotype of microsatellite instability (MSI), and most cases of FAP are caused by germline APC mutations. To date, non‐FAP familial CRC aggregates have not been evaluated for germline APC mutations. In this study, we examined the involvement of germline APC mutations in 79 individuals with CRC who had early‐age onset of their cancer (age < 50 years) and/or a family history of CRC. Cases with FAP or HNPCC due to defective mismatch repair were excluded from the study. Using conformation‐sensitive gel electrophoresis and the protein truncation test as the screening methods, no functionally significant germline mutations were detected for any of the cases. An apparently silent polymorphism resulting in a 1‐bp alteration of A → G (proline → proline) in exon 4 was observed. Additionally, four intervening sequence (IVS) alterations were detected: IVS2‐53t→c in 3 cases; IVS4‐17ins T in 3 cases; IVS5+32t→c in 16 cases; and IVS5+33g→a in 1 case. All appeared to be polymorphisms present in similar proportions in an average‐risk population. We conclude that germline APC mutations do not account for familial MSS (stable microsatellite) CRC associated with few synchronous polyps. ©2000 Wiley‐Liss, Inc.

Certification in Molecular Pathology in the United States (Training and Education Committee, the Association for Molecular Pathology)

Training in molecular pathology in the United States is undergoing development toward a more structured format involving accreditation of training programs and the availability of recognized professional credentials. The traditional apprenticeship for molecular pathology, composed of experience in a molecular biology laboratory with a strong research focus is being replaced by formal training in residency, fellowship, and other postdoctoral training programs that have a clinical focus. This is a reflection of increasing importance of this field to clinical practice, and to a growing desire of persons interested in molecular pathology to undertake formal training programs. These developments are bringing molecular pathology in line with other clinical laboratory specialties for which structured training and certification have long been the norm. An important measure of educational achievement is success in professional examinations leading to recognized credentials. These credentials should attest to the holder’s professional competence as a practitioner in the field and are frequently used for this purpose by licensing and other regulatory agencies. In the past decade, and especially in the last 5 years, a number of routes for certification in molecular pathology by examination have been offered by nationally recognized credentialing agencies. This paper provides an update on these certification routes reflective of the growing interest in molecular pathology education. The principal Boards that offer certification in the United States are the American Board of Pathology, the American Board of Medical Genetics, the American Board for Clinical Chemistry, the National Credentialing Agency for Laboratory Personnel, and the American Board of Bioanalysis. The American Board of Medical Genetics offered a combined examination in Clinical Molecular Genetics and Clinical Biochemical Genetics in 1990 and has offered an examination in Clinical Molecular Genetics alone since 1993. Because of the overlap between molecular genetics and molecular pathology, we have included this examination in this listing. The information provided here is intended to summarize the requirements for these exams. Complete requirements are available from the boards listed here.