Expanded carrier screening for inherited genetic disease using exome and genome sequencing.

The goal of this study was to assess the feasibility of using exome (ES) and genome sequencing (GS) in guiding preconception genetic screening (PCGS) for couples who are planning to conceive by creating a workflow for identifying risk alleles for autosomal recessive (AR) and X-linked (XL) disorders without the constraints of a predetermined, targeted gene panel. There were several limitations and challenges related to reporting and the technical aspects of ES and GS, which are listed in the discussion. We selected 150 couples from a cohort of families (trios) enrolled in a research protocol where the goal was to define the genetic etiology of disease in an affected child. Pre-existing, de-identified parental sequencing data were analyzed to define variants that would place the couple at risk of having a child affected by an AR or XL disorder. We identified 17 families who would be selected for counseling about risk alleles. We noted that only 3 of these at-risk couples would be identified if we limited ourselves to the current ACMG-recommended expanded carrier screening gene panel. ES and GS successfully identified couples who are at risk of having a child with a rare AR or XL disorder that would have been missed by the current recommended guidelines. Current limitations of this approach include ethical concerns, difficulties in reporting results including variant calling due to the rare nature of some of the variants, determining which disorders to report, as well as technical difficulties in detecting certain variants such as repeat expansions.

ECFS standards of care on CFTR-related disorders: Towards a comprehensive program for affected individuals.

After three publications defining an updated guidance on the diagnostic criteria for people with cystic fibrosis transmembrane conductance regulator (CFTR)-related disorders (pwCFTR-RDs), establishing its relationship to CFTR-dysfunction and describing the individual disorders, this fourth and last paper in the series addresses some critical challenges facing health care providers and pwCFTR-RD. Topics included are: 1) benefits and obstacles to collect data from pwCFTR-RD are discussed, together with the opportunity to integrate them into established CF-registries; 2) the potential of infants designated CRMS/CFSPID to develop a CFTR-RD and how to communicate this information; 3) a description of the challenges in genetic counseling, with particular regard to phenotypic variability, unknown long-term evolution, CFTR testing and pregnancy termination 4) a proposal for the assessment of potential barriers to the implementation and dissemination of the produced documents to health care professionals involved in the care of pwCFTR-RD and a process to monitor the implementation of the CFTR-RD recommendations; 5) clinical trials investigating the efficacy of CFTR modulators in CFTR-RD and how endpoints and outcomes might be adapted to the heterogeneity of these disorders.

AAV-based gene therapy prevents neuropathology and results in normal cognitive development in the hyperargininemic mouse.

Complete arginase I deficiency is the least severe urea cycle disorder, characterized by hyperargininemia and infrequent episodes of hyperammonemia. Patients suffer from neurological impairment with cortical and pyramidal tract deterioration, spasticity, loss of ambulation and seizures, and is associated with intellectual disability. In mice, onset is heralded by weight loss beginning around day 15; gait instability follows progressing to inability to stand and development of tail tremor with seizure-like activity and death. Here we report that hyperargininemic mice treated neonatally with an adeno-associated virus (AAV)-expressing arginase and followed long-term lack any presentation consistent with brain dysfunction. Behavioral and histopathological evaluation demonstrated that treated mice are indistinguishable from littermates, and that putative compounds associated with neurotoxicity are diminished. In addition, treatment results in near complete resolution of metabolic abnormalities early in life; however, there is the development of some derangement later with decline in transgene expression. Ammonium challenging revealed that treated mice are affected by exogenous loading much greater than littermates. These results demonstrate that AAV-based therapy for hyperargininemia is effective and prevents development of neurological abnormalities and cognitive dysfunction in a mouse model of hyperargininemia; however, nitrogen challenging reveals that these mice remain impaired in the handling of waste nitrogen.

Targeted massively parallel sequencing provides comprehensive genetic diagnosis for patients with disorders of sex development.

Disorders of sex development (DSD) are rare disorders in which there is discordance between chromosomal, gonadal, and phenotypic sex. Only a minority of patients clinically diagnosed with DSD obtains a molecular diagnosis, leaving a large gap in our understanding of the prevalence, management, and outcomes in affected patients. We created a novel DSD-genetic diagnostic tool, in which sex development genes are captured using RNA probes and undergo massively parallel sequencing. In the pilot group of 14 patients, we determined sex chromosome dosage, copy number variation, and gene mutations. In the patients with a known genetic diagnosis (obtained either on a clinical or research basis), this test identified the molecular cause in 100% (7/7) of patients. In patients in whom no molecular diagnosis had been made, this tool identified a genetic diagnosis in two of seven patients. Targeted sequencing of genes representing a specific spectrum of disorders can result in a higher rate of genetic diagnoses than current diagnostic approaches. Our DSD diagnostic tool provides for first time, in a single blood test, a comprehensive genetic diagnosis in patients presenting with a wide range of urogenital anomalies.

The M694V mutation in Armenian-Americans: a 10-year retrospective study of MEFV mutation testing for familial Mediterranean fever at UCLA.

Familial Mediterranean fever (FMF), inherited in an autosomal recessive manner, is a systemic auto-inflammatory disorder characterized by recurrent attacks of fever with peritonitis, pleuritis, synovitis and erysipeloid rash. The marenostrin-encoding fever (MEFV) gene, located on chromosome 16p13.3, is the only gene in which mutations are currently known to cause FMF. To correlate specific genotypes with adverse phenotypes of affected populations residing in the Western United States, a retrospective case series review was conducted of all MEFV gene mutation testing completed at UCLA Clinical Molecular Diagnostic Laboratory between February 2002 and February 2012, followed by clinical chart review of all subjects who either have a single or double mutation. All 12 common mutations in the MEFV gene were analyzed and the M694V variant was found to be associated with an adverse FMF clinical outcome in the Armenian-American population, manifested by earlier onset of disease, increased severity of disease, and renal amyloidosis.

Arginase induction by sodium phenylbutyrate in mouse tissues and human cell lines.

Hyperargininemia is a urea cycle disorder caused by mutations in the gene for arginase I (AI) resulting in elevated blood arginine and ammonia levels. Sodium phenylacetate and a precursor, sodium phenylbutyrate (NaPB) have been used to lower ammonia, conjugating glutamine to produce phenylacetylglutamine which is excreted in urine. The elevated arginine levels induce the second arginase (AII) in patient kidney and kidney tissue culture. It has been shown that NaPB increases expression of some target genes and we tested its effect on arginase induction. Eight 9-week old male mice fed on chow containing 7.5 g NaPB/kg rodent chow and drank water with 10 g NaPB/L, and four control mice had a normal diet. After one week all mice were sacrificed. The arginase specific activities for control and NaPB mice, respectively, were 38.2 and 59.4 U/mg in liver, 0.33 and 0.42 U/mg in kidney, and 0.29 and 1.19 U/mg in brain. Immunoprecipitation of arginase in each tissue with AI and AII antibodies showed the activity induced by NaPB is mostly AI. AII may also be induced in kidney. AI accounts for the fourfold increased activity in brain. In some cell lines, NaPB increased arginase activity up to fivefold depending on dose (1-5 mM) and exposure time (2-5 days); control and NaPB activities, respectively, are: erythroleukemia, HEL, 0.06 and 0.31 U/mg, and K562, 0.46 and 1.74 U/mg; embryonic kidney, HEK293, 1.98 and 3.58 U/mg; breast adenocarcinoma, MDA-MB-468, 1.11 and 4.06 U/mg; and prostate adenocarcinoma, PC-3, 0.55 and 3.20 U/mg. In MDA-MB-468 and HEK most, but not all, of the induced activity is AI. These studies suggest that NaPB may induce AI when used to treat urea cycle disorders. It is relatively less useful in AI deficiency, although it could have some effect in those patients with missense mutations.

Expression of arginase isozymes in mouse brain.

The two forms of arginase (AI and AII) in man, identical in enzymatic function, are encoded in separate genes and are expressed differentially in various tissues. AI is expressed predominantly in the liver cytosol and is thought to function primarily to detoxify ammonia as part of the urea cycle. AII, in contrast, is predominantly mitochondrial, is more widely expressed, and is thought to function primarily to produce ornithine. Ornithine is a precursor in the synthesis of proline, glutamate, and polyamines. This study was undertaken to explore the cellular and regional distribution of AI and AII expression in brain using in situ hybridization and immunohistochemistry. AI and AII were detected only in neurons and not in glial cells. AI presented stronger expression than AII, but AII was generally coexpressed with AI in most cells studied. Expression was particularly high in the cerebral cortex, cerebellum, pons, medulla, and spinal cord neurons. Glutamic acid decarboxylase 65 and glutamic acid decarboxylase 67, postulated to be related to the risk of glutamate excitotoxic and/or gamma-aminobutyric acid inhibitoxic injury, were similarly ubiquitous in their expression and generally paralleled arginase expression patterns, especially in cerebral cortex, hippocampus, cerebellum, pons, medulla, and spinal cord. This study showed that AI is expressed in the mouse brain, and more strongly than AII, and sheds light on the anatomic basis for the arginine-->ornithine-->glutamate-->GABA pathway.

Quality control in molecular genetic testing.

DNA-based testing for genetic diseases has developed from nothing into a principal part of laboratory medicine over the past 15 years. In the rush to bring these powerful new technologies into medical use, issues of quality have not always been given sufficient attention. Efforts are now being made to assess the quality of the output of genetic testing laboratories, and the results show that there is room for improvement.

Detection of bcl-2/IgH rearrangements by quantitative-competitive PCR and capillary electrophoresis.

BACKGROUND: PCR is the primary method for detecting minimal residual disease in hematologic cancers. One such gene target is the bcl-2/immunoglobulin heavy chain (IgH) translocation found in a majority of cases of follicular lymphoma. METHODS AND RESULTS: We report an accurate method for quantitative detection of the bcl-2/IgH translocation marker of follicular lymphoma in a series of patients in various stages of remission and relapse who had been treated with a combination of ifosfamide, mitoxantrone, and etoposide (MINE) chemotherapy and monoclonal anti-CD20 antibody (Rituximab). The approach uses seminested PCR followed by analysis of the products on a fluorescent capillary electrophoresis system. The quantitation of bcl-2/IgH translocation-positive cells was sensitive and reproducible, capable of detecting as few as five malignant cells out of 300,000 normal cells. CONCLUSION: Quantitative PCR enables one to monitor the kinetics of tumor reduction in patients treated with MINE chemotherapy in combination with Rituximab.

A multiethnic study of Delta32ccr5 and ccr2b-V64I allele distribution in four Los Angeles populations.

Mutant alleles of the chemokine receptors CCR5 and CCR2 affect the susceptibility to HIV infection as well as the rate of disease progression. In this article the authors report the results of a survey for presence of the common Delta32ccr5 and ccr2b-V64I mutant alleles in 472 individuals of a multiethnic cohort. Hispanic Americans had the highest observed frequency of the Delta32ccr5 allele (3.57%), whereas African Americans had a lower frequency (1.55%). The mutant allele was absent in Asian Americans and Native Americans. Thus, the Delta32ccr5 allele segregates in populations with a significant white admixture and is rare in genetically distant non-European groups. Native Americans had the highest occurrence of the ccr2b-V64I allele (31.13%), whereas African Americans, Asian Americans, and Hispanic Americans had much lower frequencies (14.36%, 11.94%, and 14.37% respectively). This mutation is probably an ancient one, occurring before the migration of the ancestors of Native Americans across the Bering Straits to the Americas. The twofold greater frequency of ccr2b-V64I in modern Native Americans probably reflects a founder effect. The observed population differences in Delta32ccr5 and ccr2b-V64I frequencies, considered together with their documented effects on sensitivity to HIV infection and rate of disease progression, have implications for HIV transmission patterns in the United States, as well as for AIDS prediction, monitoring, and treatment.

In-frame deletions of BRCA1 may define critical functional domains.

The identification of genomic rearrangements involving more than 0.5 kb of the BRCA1 gene has confirmed a more complex mutation spectrum than was initially appreciated. Genomic rearrangements in BRCA1 represent 15% of all mutations in a group of French and American breast and ovarian cancer families and 36% of all mutations in a group of Dutch families. The rearrangements described to date range in size from 510 bp to 23.8 kb, are found throughout the gene, and are most frequently attributable to homologous recombination. We describe the identification of rearrangements in two breast and ovarian cancer families that involve 3.4 and 11.5 kb of the BRCA1 gene and span multiple exons but maintain the reading frame. Both gene rearrangements appear to result from Alu-mediated homologous recombination and have been detected by using a combination of protein truncation analysis and Southern blot analysis. These rearrangements result in the loss of amino acids that lie at the carboxy-terminus of the protein and that have previously been shown to have functional significance. Because these rearrangements result in the deletion of exons but maintain the reading frame, they may provide insights into specific regions and amino acids that have functional significance for the BRCA1 protein.

Excellent tolerance of rituximab when given after mitoxantrone/cyclophosphamide: an effective and safe combination for indolent non-Hodgkin's lymphoma.

Treatment for extensive indolent lymphoma should provide optimization of efficacy while avoiding excessive toxicity. Rituximab may be an ideal agent to combine with chemotherapy because of its lack of classical myelotoxicity. In this study, 27 patients with a variety of histologies of indolent B-cell non-Hodgkin's lymphoma have been treated utilizing a novel three-drug combination. Nine patients had relapsed disease and 18 were previously untreated. Patients first received cyclophosphamide 800 mg/m(2) and mitoxantrone 8 mg/m(2) i.v. on day 1, every 3 weeks for 2 cycles. Subsequently, patients received rituximab 375 mg/m(2) followed by mitoxantrone 8 mg/m2 every 2 weeks for 4 cycles. This regimen and, in particular, the rituximab infusion were extremely well tolerated. Only two of 27 patients experienced a grade 1/2, infusion-related reaction during the first rituximab infusion. Grade 4 neutropenia was noted at some point in 16 patients who were then offered granulocyte-macrophage colony-stimulating factor support for improvement of neutropenia. No infections were noted. Alopecia was minimal. Of 27 patients, 19 achieved a complete response (CR), one achieved an unconfirmed CR (CRu), and five patients achieved a partial response, for an overall response rate of 92.5%. Molecular remissions were noted in seven of 12 tested patients in CR. We concluded that the cyclophosphamide/mitoxantrone/rituximab (CyMiR) regimen is effective and extremely well tolerated. Furthermore, rituximab infusion-associated morbidity is markedly reduced.