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Spinal muscular atrophy (SMA) is an autosomal recessive disorder predominately caused by bi-allelic loss of the SMN1 gene. Increased copies of SMN2, a low functioning nearly identical paralog, are associated with a less severe phenotype. SMA was recently recommended for inclusion in newborn screening. Clinical laboratories must accurately measure SMN1 and SMN2 copy number to identify SMA patients and carriers, and to identify individuals likely to benefit from therapeutic interventions. Having publicly available and appropriately characterized reference materials with various combinations of SMN1 and SMN2 copy number variants is critical to assure accurate SMA clinical testing. To address this need, the CDC-based Genetic Testing Reference Materials Coordination Program, in collaboration with members of the genetic testing community and the Coriell Institute for Medical Research, has characterized 15 SMA reference materials derived from publicly available cell lines. DNA samples were distributed to four volunteer testing laboratories for genotyping using three different methods. The characterized samples had zero to four copies of SMN1 and zero to five copies SMN2. The samples also contained clinically important allele combinations (eg, zero copies SMN1, three copies SMN2), and several had markers indicative of an SMA carrier. These and other reference materials characterized by the Genetic Testing Reference Materials Coordination Program are available from the Coriell Institute and are proposed to support the quality of clinical laboratory testing.

EVEN-PLUS syndrome is a rare condition characterized by its involvement of the Epiphyses, Vertebrae, Ears, and Nose, PLUS other associated findings. We report here the fifth case of EVEN-PLUS syndrome with novel variants c.818 T > G (p.L273X) and c.955C > T (p.L319F) in the HSPA9 gene identified through whole-exome sequencing. The patient is the first male known to be affected and presented with additional features not previously described with EVEN-PLUS syndrome. These features include agenesis of the septum pellucidum, a short chest and sternum, 13 pairs of ribs, a single hemivertebra, laterally displaced nipples, hydronephrosis, unilateral cryptorchidism, unilateral single palmar crease, bilateral clubfoot, and hypotonia. qPCR analysis provides supporting evidence for a nonsense-mediated decay mechanism for the HSPA9 truncating variant. In silico 3D modeling supports the pathogenicity of the c.955C > T (p.L319F) missense variant. The study presented here further describes the syndrome and broadens its mutational and phenotypic spectrum. Our study also lends support to HSPA9 variants as the underlying etiology of EVEN-PLUS syndrome and ultimately provides a better understanding of the molecular basis of the condition.

PURPOSE Precision medicine approaches, including germline pharmacogenetics (PGx) and management of drug-drug interactions (DDIs), are likely to benefit patients with advanced cancer who are frequently prescribed multiple concomitant medications to treat cancer and associated conditions. Our objective was to assess the potential opportunities for PGx and DDI management within a cohort of adults with advanced cancer. METHODS Medication data were collected from the electronic health records for 481 subjects since their first cancer diagnosis. All subjects were genotyped for variants with clinically actionable recommendations in Clinical Pharmacogenetics Implementation Consortium guidelines for 13 pharmacogenes. DDIs were defined as concomitant prescription of strong inhibitors or inducers with sensitive substrates of the same drug-metabolizing enzyme and were assessed for six major cytochrome P450 (CYP) enzymes. RESULTS Approximately 60% of subjects were prescribed at least one medication with Clinical Pharmacogenetics Implementation Consortium recommendations, and approximately 14% of subjects had an instance for actionable PGx, defined as a prescription for a drug in a subject with an actionable genotype. The overall subject-level prevalence of DDIs and serious DDIs were 50.3% and 34.8%, respectively. Serious DDIs were most common for CYP3A, CYP2D6, and CYP2C19, occurring in 24.9%, 16.8%, and 11.7% of subjects, respectively. When assessing PGx and DDIs together, approximately 40% of subjects had at least one opportunity for a precision medicine–based intervention and approximately 98% of subjects had an actionable phenotype for at least one CYP enzyme. CONCLUSION Our findings demonstrate numerous clinical opportunities for germline PGx and DDI management in adults with advanced cancer.

BackgroundPatients with chronic kidney disease (CKD) often have uncontrolled hypertension despite polypharmacy. Pharmacogenomic drug-gene interactions (DGIs) may impact the metabolism or efficacy of antihypertensive agents. We hypothesized that providing a panel of 11 pharmacogenomic predictors of antihypertensive response would improve hypertension control.MethodsA prospective cohort with CKD and hypertension was followed to assess the effect of pharmacogenomic testing on blood pressure control. The analysis population included 382 hypertensive subjects genotyped for cross-sectional assessment of drug-gene interactions and 335 subjects followed for 1 year to assess systolic (SBP) and diastolic blood pressure (DBP).ResultsMost participants (58.2%) with uncontrolled hypertension had a DGI reducing the efficacy of one or more antihypertensive agents. Subjects with a DGI had 1.88-fold (95% CI 1.2-2.8) higher odds of uncontrolled hypertension as compared to those without a DGI, adjusted for race and CKD grade. CYP2C9 reduced metabolism genotypes were associated with losartan response and uncontrolled hypertension (Odds Ratio 5.2, CI 1.9 -14.7). CYP2D6 intermediate or poor metabolizers had less frequent uncontrolled hypertension compared to normal metabolizers taking metoprolol or carvedilol (OR 0.55, CI 0.3-0.95). In 335 subjects completing 1 year follow-up, SBP (−4.0 mmHg, CI 1.6-6.5) and DBP (−3.3 mmHg, CI 2.0-4.6) were improved. The magnitude of reductions in SBP (−14.8 mmHg, CI 10.3-19.3) and DBP (−8.4 mmHg, CI 5.9-10.9) were greatest in the 90 individuals with uncontrolled hypertension and an actionable genotype.ConclusionsThere is a potential role for the addition of pharmacogenomic testing to optimize antihypertensive regimens in patients with CKD.

A precision health initiative was implemented across a multi-hospital health system, wherein a panel of genetic variants was tested and utilized in the clinical care of chronic kidney disease (CKD) patients. Pharmacogenomic predictors of antihypertensive response and genomic predictors of CKD were provided to clinicians caring for nephrology patients. To assess clinician knowledge, attitudes, and willingness to act on genetic testing results, a Likert-scale survey was sent to and self-administered by these nephrology providers (N = 76). Most respondents agreed that utilizing pharmacogenomic-guided antihypertensive prescribing is valuable (4.0 &plusmn, 0.7 on a scale of 1 to 5, where 5 indicates strong agreement). However, the respondents also expressed reluctance to use genetic testing for CKD risk stratification due to a perceived lack of supporting evidence (3.2 &plusmn, 0.9). Exploratory sub-group analyses associated this reluctance with negative responses to both knowledge and attitude discipline questions, thus suggesting reduced exposure to and comfort with genetic information. Given the evolving nature of genomic implementation in clinical care, further education is warranted to help overcome these perception barriers.

Aims: To validate a laboratory-developed test for the nucleoside transporter, SLC28A3, which has been associated with an increased risk of anthracycline-induced cardiomyopathy. Methods: We used Taqman(®) allele discrimination to test for two variants of the SLC28A3 gene: rs7853758 (c.1381C>T) and rs885004 (c.862-360C>T). Results: During the validation process, we noted that several DNA samples obtained from the Coriell Cell Repository (Camden, NJ) were positive for both the c.1381 C > T and c.862-360C>T variants and another variant allele for either c.1381 C > T or c.862-360C>T (e.g., c.1381C>T homozygous/c.862-360C>T heterozygous, c.1381C>T homozygous/c.862-360C>T homozygous). We used de-identified DNA samples from trios of family members (mother, father, and child) to establish that the c.1381 C > T and c.862-360C>T variant alleles could be inherited in cis on the same chromosome. Conclusions: Samples containing three variant alleles suggest that the c.1381 C > T and c.862-360C>T are in cis on the chromosome in some individuals and may have implications when calculating anthracycline-induced cardiomyopathy risk. In this study, we confirm a novel haplotype of SLC28A3 using familial studies.

Coffin-Lowry syndrome (CLS) is a rare X-linked disorder characterized by moderate to severe intellectual disability, hypotonia, craniofacial features, tapering digits, short stature, and skeletal deformities. Using whole exome sequencing and high-resolution targeted comparative genomic hybridization array analysis, we identified a novel microduplication encompassing exons five through nine of RPS6KA3 in three full brothers. Each brother presented with intellectual disability and clinical and radiographic features consistent with CLS. qRT-PCR analyses performed on mRNA from the peripheral blood of the three siblings revealed a marked reduction of RPS6KA3 levels suggesting a loss-of-function mechanism. PCR analysis of the patients' cDNA detected a band greater than expected for an exon 4-10 amplicon, suggesting this was likely a direct duplication that lies between exons 4 through 10, which was later confirmed by Sanger sequencing. This microduplication is only the third intragenic duplication of RPS6KA3, and the second and smallest reported to date thought to cause CLS. Our study further supports the clinical utility of methods such as next-generation sequencing and high-resolution genomic arrays to detect small intragenic duplications. These methods, coupled with expression studies and cDNA structural analysis have the capacity to confirm the diagnosis of CLS in these rare cases.

Hypertension and chronic kidney disease are inextricably linked. Hypertension is a well-recognized contributor to chronic kidney disease progression and, in turn, renal disease potentiates hypertension. A generalized approach to drug selection and dosage has not proven effective in managing these conditions, in part, because patients with heterogeneous kidney disease and hypertension etiologies are frequently grouped according to functional or severity classifications. Genetic testing may serve as an important tool in the armamentarium of clinicians who embrace precision medicine. Increasing scientific evidence has supported the utilization of genomic information to select efficacious antihypertensive therapy and understand hereditary contributors to chronic kidney disease progression. Given the wide array of antihypertensive agents available and diversity of genetic renal disease predictors, a panel-based approach to genotyping may be an efficient and economic means of establishing an individualized blood pressure response profile for patients with various forms of chronic kidney disease and hypertension. In this manuscript, we discuss the validation process of a Clinical Laboratory Improvement Amendments (CLIA)-approved genetic test to relay information on 72 genetic variants associated with kidney disease progression and hypertension therapy. These genomic-based interventions, in addition to routine clinical data, may help inform physicians to provide personalized therapy.

Methylation of CpG islands in gene promoter regions is a major molecular mechanism of gene silencing and underlies both cancer development and progression. In molecular oncology, testing for the CpG methylation of tissue DNA has emerged as a clinically useful tool for tumor detection, outcome prediction, and treatment selection, as well as for assessing the efficacy of treatment with the use of demethylating agents and monitoring for tumor recurrence. In addition, because CpG methylation occurs early in pre-neoplastic tissues, methylation tests may be useful as markers of cancer risk in patients with either infectious or inflammatory conditions. The Methylation Working Group of the Clinical Practice Committee of the Association of Molecular Pathology has reviewed the current state of clinical testing in this area. We report here our summary of both the advantages and disadvantages of various methods, as well as the needs for standardization and reporting. We then conclude by summarizing the most promising areas for future clinical testing in cancer molecular diagnostics.

The number of different laboratories that perform genetic testing for cystic fibrosis is increasing. However, there are a limited number of quality control and other reference materials available, none of which cover all of the alleles included in commercially available reagents or platforms. The alleles in many publicly available cell lines that could serve as reference materials have neither been confirmed nor characterized. The Centers for Disease Control and Prevention-based Genetic Testing Reference Material Coordination Program, in collaboration with members of the genetic testing community as well as Coriell Cell Repositories, have characterized an extended panel of publicly available genomic DNA samples that could serve as reference materials for cystic fibrosis testing. Six cell lines [containing the following mutations: E60X (c.178G>T), 444delA (c.312delA), G178R (c.532G>C), 1812–1G>A (c.1680–1G>A), P574H (c.1721C>A), Y1092X (c.3277C>A), and M1101K (c.3302T>A)] were selected from those existing at Coriell, and seven [containing the following mutations: R75X (c.223C>T), R347H (c.1040G>A), 3876delA (c.3744delA), S549R (c.1646A>C), S549N (c.1647G>A), 3905insT (c.3773_3774insT), and I507V (c.1519A>G)] were created. The alleles in these materials were confirmed by testing in six different volunteer laboratories. These genomic DNA reference materials will be useful for quality assurance, proficiency testing, test development, and research and should help to assure the accuracy of cystic fibrosis genetic testing in the future. The reference materials described in this study are all currently available from Coriell Cell Repositories.

Cystic fibrosis population carrier screening: 2004 revision of American College of Medical Genetics mutation panel

The US Food and Drug Administration (FDA) notified Congress July 31, 2014,1 of its intent to regulate laboratory developed tests. These encompass thousands of clinical assays currently used in medical practice including most pharmacogenetic tests. This guidance has the potential to impact the innovation and sustainability of pharmacogenetic research and its clinical implementation. We anticipate that several requirements could have substantial implications for both clinical research and the clinical implementation of pharmacogenetics. Input from federal funding agencies (eg, National Institutes of Health, Agency for Health Research Quality, Patient-Centered Outcomes Research Institute) will be needed to clarify who will bear the financial burden of some of the increased requirements. Further, key clarifications will be required from the FDA to understand how implementation will occur over the next decade (Figure 1). Clarity now will allow the research and clinical communities to effectively plan and budget for these changes. Below, we identify and describe the key issues. Figure 1 FDA timeline for implementation of oversight of laboratory-developed tests. *Time from FDA comment review and congressional input to the release of the final guidance is unknown. **PMA process is based on a per se demonstration of safety and effectiveness ... Over the past decades, many pharmacogenomics biomarkers that are responsible for clinical variability in the pharmacokinetics and pharmacodynamics of drug therapy have been identified. These studies have mostly been conducted using laboratory-developed procedures or tests (LDTs).2 According to the published guidance, it appears that these clinical discovery and validation studies will now need to obtain investigational device exemptions (IDEs) from the FDA. For example, conducting a simple clinical study to determine if genetic variants in a drug-metabolizing enzyme are associated with the pharmacokinetics of a drug would require that the genotyping assay laboratory obtain an IDE before conducting the study. IDE submissions require substantial expertise and resources that are not typically common in academic research centers or Clinical Laboratory Improvement Amendment (CLIA)–certified clinical laboratories. This may even be new for drug companies that may have expertise in new drug application submissions, but not IDEs. Therefore, it will substantially increase the cost and resources needed to conduct these studies. It is not clear to us what harm has been done that will be solved by the IDE requirement that justifies such a change. The question of funding is important. Will funding agencies be willing to support these additional costs, or will it just reduce the scope or number of studies that will be able to be conducted? We suggest that the FDA use its enforcement discretion and not require IDEs for these types of translational studies. Based on the language in the guidance document, clinical (ie, CLIA) laboratories providing pharmacogenetic genotyping using an LDT will be required to obtain premarket approvals (PMAs). As there are very few FDA-approved pharmacogenetic in vitro diagnostic devices, most clinical testing using LDTs is performed in CLIA laboratories, often with College of American Pathologists accreditation. The time and costs involved in obtaining PMAs are high. In a 2010 survey and analysis conducted by AdvaMed,3 US patients waited an average of 2 years longer than those in Europe to gain access to new medical technologies, which was attributable to the FDA device approval. In addition, the average total cost to bring a low to moderate in vitro diagnostic through the FDA 510(k) process was $31 million, with $24 million spent on FDA-dependent or -related activities. For higher-risk products submitted for approval, the average cost increased to $94 million, with $75 million linked to FDA requirements. In addition, the industry reported an average time from first communication with the FDA to clearance or approval ranging from 31 to 54 months. The FDA claims that CLIA certification of a laboratory performing an LDT does not provide the safety, oversight, and adverse event reporting requirements that exist with devices cleared or approved by the agency; however, this has not been supported by statistically valid evidence that this has caused harm to patients. At least publicly, they have only cited antidotal cases where a patient received inappropriate therapy as a result of a faulty LDT. In the FDA's notice to Congress of its intent to initiate oversight of LDTs (Federal Register/Vol. 79, No. 192/Friday, October 3, 2014), there was no data submitted that supports the claim that the status quo condition was causing harm. Despite this lack of evidence, it does express a concern about the potential risk to patients if LDTs do not receive oversight. The FDA in their October 2014 filing in the Federal Register noted that the complexity and expanded use of LDTs today are significantly different from 1976 when the Congress enacted Medical Device Amendment (MDA) and expanded the FDA oversight of medical devices intended for human use. To support the oversight of LDTs, the FDA is using the 1976 MDA in attempt to redefine laboratories that perform LDTs as manufacturers of medical devices intended for human use. There are potentially many implications, including financial and health economics, if the FDA is allowed to categorize these laboratories as manufacturers. During the September 9, 2014 House Committee on Energy and Commerce Hearing on the Proposed FDA Laboratory Developed Test Policy, Dr. Jeff Shuren, the Director of the Center for Devices and Radiological Health at the FDA, stated that the “FDA has not conducted a formal economic analysis” to justify the increased time and cost required to implement the proposed framework. In this context, it would be valuable to consider a “fast track” approval process or other creative approaches for new tests that have evidence of life-saving benefits (i.e., HLA-B*57:01 and abacavir), in the same way as drugs has occurred for drug approvals. Currently, many laboratories decide on a genotyping platform that fits the needs of their laboratory and then design LDTs for multiple assays on a single platform. However, with companion diagnostics in which the drug and device are approved together by the FDA, drug and device manufacturers essentially have 1 instrument per drug, making it necessary for the laboratories to obtain multiple different instruments to provide pharmacogenomic services for multiple drugs. The initial capital investment and routine running costs (eg, maintenance contracts, training, and competency) will thus be much higher for laboratories. Depending on the device labeling, it could even require different instruments for the same genetic test when used for different drugs (eg, CYP2D6 testing for codeine and antidepressants), even though they test the exact same genetic variants. If the device (ie, instrument and test kit) is used for a different intended use (ie, same gene, different drug), then the laboratory will need to submit to the FDA to demonstrate cross-platform equivalency. Last, input from payers will be needed to understand how reimbursement for clinical testing will be handled. For example, once a test is FDA-approved or -cleared, will the payers reimburse those tests at a significantly higher rate, so the clinical laboratory can recoup its cost for the FDA submission? The current procedural terminology coding system for reimbursement is platform agnostic. Though Centers for Medicare and Medicaid Service has a pilot program to differentiate reimbursement based on platform,4 will the payers even allow reimbursement for non-FDA-approved tests when there are FDA-approved tests available? In conclusion, many questions remain to be answered related to the new FDA guidance on LDTs with respect to clinical research and implementation of pharmacogenetic testing. The FDA should provide specific evidence about the harm caused by LDTs, so a less obstructive solution could be identified. Although it may be true that there are some CLIA-accredited laboratories that have developed their own methods and tests that do not meet acceptable quality standards, why not selectively pursue these entities through existing or enhanced CLIA regulations rather than imposing FDA-directed oppressive and costly guidance across the entire spectrum of laboratory service providers and researchers?

We report a G-->A transition at nucleotide 431 of the proteolipid protein gene (PLP) results in a nonsense codon in a family with an unusual form of Pelizaeus-Merzbacher disease (PMD). The mutation, which creates a second AluI restriction site, results in a nonsense mutation in PLP. The clinical picture resembles somewhat that of X-linked spastic paraplegia (SPG). It differs from this and both the classical and connatal forms of PMD in that it is relatively mild in form, onset is delayed beyond age 2 years, nystagmus is absent, tremors are prominent, mental retardation is not severe, some patients show dementia or personality disorders, the disease is progressive rather than static in some, and several females show signs of disease. The nonsense mutation, which is in exon 3B, should block the synthesis of normal PLP but spare DM20, the isoform whose persistence has been associated with mild forms of PLP-associated disease in both humans and mice.

We studied a female infant with clinical signs of Pelizaeus-Merzbacher disease (PMD), who has a familial mutation (C41-->T) in exon 2 of the proteolipid protein gene (PLP), and selected relatives. While the carrier mother and grandmother of the proposita currently are neurologically normal and show normal T2 magnetic resonance imaging (MRI) of the brain, the infant has a neurological picture, MRIs, and brain auditory evoked response (BAER) consistent with that diagnosis. The data here presented show that PMD can occur in females carrying a mutation in the PLP gene. Our experience with the MRIs of this patient, her mother and grandmother, and those of a previously reported family [Pratt et al.: Am J Med Genet 38:136-139, 1991] show that molecular genetic analysis and not MRI is the appropriate means for carrier detection.

Angelman and Prader-Willi syndromes are reciprocal imprinting disorders caused by loss of maternally or paternally expressed genes, respectively, within 15q11.2-q13. Angelman syndrome (AS; OMIM 105830) is a neurodevelopmental disorder and is due to the loss of maternally expressed UBE3A gene. Prader-Willi syndrome (PWS; OMIM 176270) is a clinically distinct disorder caused by the loss of paternally expressed genes in the human chromosome region 15q11.2-q13. Recently published data strongly suggest a role for the paternally expressed small nucleolar RNA (snoRNA) cluster, SNORD116, in PWS etiology. Uniparental disomy (UPD) 15 is one of the important causes of PWS and AS. Interestingly, balanced and unbalanced chromosomal aberrations in the form of Robertsonian translocation, isochromosomes, supernumerary marker chromosomes and copy number variations have been strongly linked with the occurrence of UPD. Here we report on a very unique case with a mosaic isochromosome for the entire long arm of 15, that is, i(15)(q10), resulting in mosaic uniparental isodisomy for 15q and with no copy number alterations. This is the first report of UPD15 constituted by a mosaic, but copy number neutral chromosomal rearrangement in a patient with a variant PWS-like phenotype.

Tamoxifen, a widely prescribed drug for the treatment and prevention of breast cancer, is metabolized to more potent metabolites by the cytochrome P450 2D6 (CYP2D6) enzyme. Variants in the CYP2D6 gene can cause patients to be either intermediate or poor metabolizers, thereby rendering tamoxifen treatment less effective. Testing for CYP2D6 gene variants is available in Clinical Laboratory Improvement Amendments–certified clinical laboratories; however, the biological complexity of the variants makes result interpretation and phenotype prediction challenging. This article describes the clinical significance of variants as well as important analytical, interpretative, and reporting issues. It is designed to be a guideline for clinical laboratory professionals in performing tests and interpreting results with respect to CYP2D6 genetic variants. Genet Med 2012:14(12):990–1000

Cystic fibrosis [(CF); Online Mendelian Inheritance in Man No. 602421] is an autosomal recessive disorder characterized by chronic broncho-pulmonary disease, pancreatic insufficiency, and increased sweat electrolytes (1). The CF transmembrane conductance regulator ( CFTR ) gene was identified by three groups (2)(3)(4) and was mapped to the long arm of chromosome 7 (7q31). The CFTR contains 24 exons, encodes a protein of 1480 amino acids, functions as a chloride channel, and regulates other transport pathways. Over 900 disease-causing mutations in the CFTR gene have been identified. The American College of Medical Genetics has recommended a core mutation panel for general population CF carrier screening (5). This panel contains 25 of the known CF mutations, including 3120+1G→A and I148T . 3120+1G→A (intron 16) is the second most common CF mutation in the African American population and accounts for 9–14% of the African American CF chromosomes (6). I148T (exon 4) is the second most common CF mutation in the French Canadian population and accounts for 9.1% of the French Canadian CF chromosomes (7). Most of the 25 mutations recommended by the American College of Medical Genetics can be detected with commercially available methods [Roche Molecular Biochemicals, Applied Biosystems and Innogenetics (ABI)], but no assays test for both the 3120+1G→A and I148T mutations, and two of the three assays do not detect either mutation. Therefore, we developed a multiplex primer-extension assay that rapidly and simultaneously detects these two CF mutations using the ABI Prism® SNaPshotTM ddNTP Primer-Extension assay. We believe this is the first demonstration of the use of this assay in the literature. The method uses a dideoxynucleotide primer-extension reaction (PER) in conjunction with an ABI genetic analyzer to detect the addition of a fluorescently labeled ddNTP to the 3′ end of a sequencing primer (Fig. …

The purpose of this work is to define rare variants of cystic fibrosis (CF) that are potential sources of error and can confound molecular genetic testing methods. We performed routine, clinical CF mutation screening using a laboratory-developed test and the oligonucleotide ligation assay reagents from Abbott/Celera. In this report, we describe 11 unique allele drop outs [3849 + 10kb C>T (NM_000492.2:c.3718-2477C>T), V520F (c.1558G>T), 1078delT (c.948delT), A455E (c.1364C>A), R347P (c.1040G>C), 2184delA (c.2052delA), W1282X (c.3846G>A), R117H (c.350G>A), G85E (c.254G>A), 621 + 1G>T (c.489 + 1G>T), and 2789 + 5G>A (c.2657 + 5G>A)] observed with this platform. The allele drop outs account for less than 0.01% of all results reported in our laboratory. Both the recognition and enumeration of such variants along with clinical information in CF testing is valuable in avoiding false-positive and false-negative results.

Fragile X syndrome, which is caused by expansion of a (CGG)(n) repeat in the FMR1 gene, occurs in approximately 1:3500 males and causes mental retardation/behavioral problems. Smaller (CGG)(n) repeat expansions in FMR1, premutations, are associated with premature ovarian failure and fragile X-associated tremor/ataxia syndrome. An FMR1-sizing assay is technically challenging because of high GC content of the (CGG)(n) repeat, the size limitations of conventional PCR, and a lack of reference materials available for test development/validation and routine quality control. The Centers for Disease Control and Prevention and the Association for Molecular Pathology, together with the genetic testing community, have addressed the need for characterized fragile X mutation reference materials by developing characterized DNA samples from 16 cell lines with repeat lengths representing important phenotypic classes and diagnostic cutoffs. The alleles in these materials were characterized by consensus analysis in nine clinical laboratories. The information generated from this study is available on the Centers for Disease Control and Prevention and Coriell Cell Repositories websites. DNA purified from these cell lines is available to the genetics community through the Coriell Cell Repositories. The public availability of these reference materials should help support accurate clinical fragile X syndrome testing.

Purpose: Genotyping 37,026 individuals as part of a thrombophilia evaluation, we determined and analyzed the genotypic frequencies of the 677CT and 1298AC mutations in the methylenetetrahydrofolate reductase (MTHFR) gene. Methods: The 677CT and 1298AC mutations in the MTHFR gene were determined by either a laboratory-developed test involving PCR amplification and restriction digestion utilizing the ABI 3100 capillary electrophoresis apparatus (Applied Biosystems Inc) or by using an Analyte Specific Reagent (ASR) supplied by Third Wave Technologies. The genotype for three specimens with triple variant MTHFR mutations were confirmed by DNA sequencing on the ABI 3100 capillary electrophoresis apparatus. Results: The MTHFR frequencies of the 677CT/1298AA, 677CC/1298AC, 677CT/1298AC, 677CC/1298AA, 677TT/1298AA, 677CC/1298CC, 677TT/1298AC, and 677CT/ 1298CC genotypes were 0.228, 0.208, 0.198, 0.153, 0.122, 0.088, 0.0005, and 0.0003, respectively. Conclusions: Individuals containing double variant MTHFR mutations on one allele (cis) cannot be distinguished between compound heterozygotes (trans) for 677CT and 1298AC mutations in routine clinical testing, a genotype associated with thrombophilia. Such patients could be inappropriately counseled for being at high risk for thrombotic episodes. Until information regarding prevalence and the clinical consequences of this double variant (cis) allele becomes available, caution should be used in interpreting the genotyping results of compound heterozygosity for 677CT and 1298AC.

A 23-year-old man with Pelizaeus-Merzbacher disease had a novel mutation, C344A (Thr115Lys), in exon 3 of the proteolipid protein gene (PLP). His mother, heterozygous for the mutation, developed progressive personality change and a gait disorder in her mid-20s. Her MRI at age 53 showed a diffuse severe leukodystrophy. This report extends the phenotypic range of disease due to PLP gene mutations to include adult-onset dementia in females.NEUROLOGY 1996;47: 1333-1335

Purpose: We expect that the mutation panel currently recommended for preconception/prenatal CF carrier screening will be modified as new information is learned regarding the phenotype associated with specific mutations and allele frequencies in various populations. One such example is the I148T mutation, originally described as a severe CF mutation. After implementation of CF population-based carrier screening, we learned that I148T exists as a complex allele with 3199del6 in patients with clinical CF, whereas asymptomatic compound heterozygotes for I148T and a second severe CF mutation were negative for 3199del6. Methods: We performed reflex testing for 3199del6 on 663 unrelated specimens, including I148T heterozygotes, compound heterozygotes, and a homozygous individual. Results: Less than 1% of I148T carriers were also positive for 3199del6. Excluding subjects tested because of a suspected or known CF diagnosis or positive family history, 0.6% of I148T-positive individuals were also positive for 3199del6. We identified 1 I148T homozygote and 6 unrelated compound heterozygous individuals with I148T and a second CF variant (2 of whom also carried 3199del6). In addition, one fetus with echogenic bowel and one infertile male were heterozygous for I148T (3199del6 negative). Conclusions: Reflex testing for 3199del6 should be considered whenever I148T is identified. Reflex testing is of particular importance for any symptomatic patient or whenever one member of a couple carries a deleterious CF mutation and the other member is an I148T heterozygote. Further population data are required to determine if I148T, in the absence of 3199del6, is associated with mild or atypical CF or male infertility. Genet Med 2004:6(5):421–425.

We describe an in-frame deletion of parts of exons 3 and 4 of the proteolipid protein gene (PLP), with all of the intervening sequence, in a 3-generation family with Pelizaeus-Merzbacher disease. The mutation removes 49 amino acids of the PLP. © 1995 Wiley-Liss, Inc.

Single-strand conformational polymorphism analysis of an affected male with Pelizaeus-Merzbacher disease (PMD) showed a slight change in mobility of amplified exon 5 of the proteolipid protein (PLP) gene. The exon was sequenced and a G A transition at codon 216 was found. This mutation eliminates a BstNI restriction site and creates a MaeI restriction site. In 1989, Gencic et al. reported a mutation that destroyed the same BstNI site, but resulted in a substitution at codon 215 [Am J Hum Genet 45:435–442]. The mutation we report here is also present in the patient's mother and her male fetus as determined by polymerase chain reaction analysis of amniocytes. © 1995 Wiley-Liss, Inc.

Context.—Bioelectronic sensors, which combine microchip and biological components, are an emerging technology in clinical diagnostic testing. An electronic detection platform using DNA biochip technology (eSensor) is under development for molecular diagnostic applications. Owing to the novelty of these devices, demonstrations of their successful use in practical diagnostic applications are limited. Objective.—To assess the performance of the eSensor bioelectronic method in the validation of 6 Epstein-Barr virus–transformed blood lymphocyte cell lines with clinically important mutations for use as sources of genetic material for positive controls in clinical molecular genetic testing. Two cell lines carry mutations in the CFTR gene (cystic fibrosis), and 4 carry mutations in the HFE gene (hereditary hemochromatosis). Design.—Samples from each cell line were sent for genotype determination to 6 different molecular genetic testing facilities, including the laboratory developing the DNA biochips. In addition to the bioelectronic method, at least 3 different molecular diagnostic methods were used in the analysis of each cell line. Detailed data were collected from the DNA biochip output, and the genetic results were compared with those obtained using the more established methods. Results.—We report the successful use of 2 applications of the bioelectronic platform, one for detection of CFTR mutations and the other for detection of HFE mutations. In all cases, the results obtained with the DNA biochip were in concordance with those reported for the other methods. Electronic signal output from the DNA biochips clearly differentiated between mutated and wild-type alleles. This is the first report of the use of the cystic fibrosis detection platform. Conclusions.—Bioelectronic sensors for the detection of disease-causing mutations performed well when used in a “real-life” situation, in this case, a validation study of positive control blood lymphocyte cell lines with mutations of public health importance. This study illustrates the practical potential of emerging bioelectronic DNA detection technologies for use in current molecular diagnostic applications.

Pelizaeus-Merzbacher disease has been known since 1885. It is characterized by severe dysmyelination of the central nervous system. We describe a new mutation in exon 6 of the proteolipid protein gene in a 9-year-old boy with severe connatal Pelizaeus-Merzbacher disease.

Alternative products of the proteolipid protein gene (PLP), proteolipid protein (PLP) and DM20, are ma- jor components of compact myelin in the central nervous system, but quantitatively minor constituents of Schwann cells. A family with a null allele of PLP has a less severe CNS phenotype than those with other types of PLP mutations. Moreover, individuals with PLP null mutations have a demyelinating peripheral neuropathy, not seen with other PLP mutations of humans or animals. Direct analysis of normal peripheral nerve demonstrates that PLP is localized to compact myelin. This and the clinical and pathologic observations of the PLP null phenotype indicate that PLP/DM20 is necessary for proper myelin function both in the central and peripheral nervous systems.

Pelizaeus-Merzbacher disease (PMD) is an X-linked dysmyelinating disorder of the central nervous system. Many cases of PMD can be attributed to defects in the proteolipid protein gene (PLP). To date, with one exception, each family has had either no or a unique mutation in one of the seven exons of PLP. We describe a new missense mutation in exon 2 of the PLP gene of an affected individual. This mutation codes for Ile instead of Thr at codon 42. The point mutation originated in the X chromosome of the maternal great-grandfather of the propositus. This was determined from the pattern of inheritance of the AhaII polymorphism and a series of microsatellite markers that are localized near PLP at Xq22.

Pelizaeus-Merzbacher disease (PMD) has been recognized as a clinical entity for more than a century. It has gradually become apparent that the disorder is a dysmyelination, in distinction to demyelinating conditions such as adrenoleukodystrophy. The failure to deposit myelin is due to decreased production of its chief protein, proteolipid protein (PLP). In about 30% of patients with the diagnosis of PMD there is a mutation in the coding portion of the proteolipid protein gene, PLP. This gene is located at Xq22 so the disease in these families shows an X-linked pattern of inheritance. The expression of the mutant gene is generally recessive, but some mutations are expressed frequently in females. At least some patients with PMD that do not show mutations in the coding region of PLP demonstrate linkage between the disease and PLP. As additional mutations in PLP are discovered, it is becoming apparent that the nosology of PLP-associated disease is changing. PMD now comprises a spectrum of disorders with similar but not necessarily identical clinical pictures. Some of these disorders may be certain forms of X-linked paraplegia, SPG2. Finally, some diseases that look like PMD may not be X-linked.

A C--greater than G transversion has been found in exon 3 of the PLP gene of affected males and their mother in a single sibship with Pelizaeus-merzbacher disease (PMD). The transversion should not result in an amino acid change in the protein but it does result in the loss of a HaeIII restriction endonuclease cleavage site. It is concordant with the disease in this family. One-hundred-ten unrelated X chromosomes are negative for this mutation. No other sequence defect was found in the PLP exons of the affected males. The cause of disease in this family remains unknown, but the association between this rare mutation and PMD is intriguing. The mutation can serve as a marker for following segregation of the PLP gene.

A C-to-T transition in exon 4 of the PLP gene was found in 2 affected males and two obligate carriers in a German family with Pelizaeus-Merzbacher disease. The mutation, which causes loss of an HphI site and changes amino acid 155 from threonine to isoleucine, was absent from 108 normal chromosomes. There are 5 concordances and 1 discrepancy between these results and those obtained by magnetic resonance imaging in this family.

While Barts hydrops is predicted when all 4 α globin genes are absent or non functional, hydrops has rarely been described with a hemoglobin H (Hb H) genotype. Hb H disease has a varied hematologic phenotype ranging from asymptomatic to transfusion dependant. The severest clinical expression is non immune hydrops and may result in fetal demise. The commonest form of Hb H disease is deletional-type. This occurs when there is deletion of α2 and α1 genes in cis and deletion of either α2 or α1 in trans. The less frequent type, but clinically more severe, is non-deletional. This is usually due to a mutation in either α2 or α1 in cis and a deletion of both α2 and α1 in trans. There are rare reports of non-deletional Hb H due to homozygous α2 mutations without any α1 deletions or mutations. Heretofore, there have been no reports of hydrops with homozygous hemoglobin Constant Spring (Hb CS). A 31-year-old Laotian woman presented at 22 weeks with polyhydramnios. Besides this, the pregnancy was uneventful for the mother. On ultrasound the fetus had a pericardial effusion and signs of congestive heart failure. The hematological and genotypic parameters of the parents and fetus are outlined in table 1. Intrauterine transfusions were performed at 22 and 29 weeks. A male infant was born vaginally at 36 weeks gestation weighing 2.3 kg. The hemoglobin was 15.9 g/dL and bilirubin was 8.4mg/dL. There was no evidence of maternofetal alloimmunization. G6PD screen was negative. By 4 weeks post-partum, the bilirubin had decreased to 0.9 mg/dL but the hemoglobin had progressively fallen to 6.9 g/dL, requiring transfusion. He had two additional transfusions at 6 and 8 weeks of life but has subsequently maintained a hemoglobin above 10 g/dL and has steadily gained weight. As in this patient, the genotypes of the other reported cases of Hb H hydrops are usually associated with a thalassemia intermedia phenotype. Thus the α globin genotypes alone do not explain why hydrops occurs. This suggests that there may be unknown genetic and/or acquired modifiers contributing to the severity of Hb H disease that results in hydrops as in this fetus with homozygous Hb CS. [Table 1] . Mother Father Fetus at 22 weeks Hemoglobin g/dL 10.0 12.5 5.2 MCV fL 86.3 81.8 143.8 MCH pg 26.5 25.6.8 46.4 RDW % 16.7 13.6 21.4 Reticulocyte % 9.9 ND 19.0 NRBC/100WBC 1 0 4756 Hemoglobin Fractions by HPLC Hb A 73.3% 97.8% 3.4% Hb F 0.8% 0.0% 77.7% Hb A2 0.9% 2.1% 0.0% Barts 0.0% 0.0% 17.7% Hb Constant Spring 2.8% 0.1% 1.2% Hb E 22.2% 0.0% 0.0% αGlobin Gene Dosage Assay by Linear PCR 4 4 4 αGlobin Gene Deletions by PCR - 3.7, - 4.2, _SEA, - 20.5, _MED,_FIL,_THAI None detected None detected None detected α2 Globin Gene Sequencing α-2CS/α-2CS α-2CS/WTα-2CS/α-2CS α1 Globin Gene Sequencing WT/WT WT/WT WT/WT βGlobin Gene Sequencing Heterozygous Hb E WT/WTWT/WT E