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.

Differential expression of the two human arginase genes in hyperargininemia. Enzymatic, pathologic, and molecular analysis.

Previous studies in our laboratory and others have demonstrated in humans and other mammals two isozymes of arginase (AI and AII) that differ both electrophoretically and antigenically. AI, a cytosolic protein found predominantly in liver and red blood cells, is believed to be chiefly responsible for ureagenesis and is the one missing in hyperargininemic patients. Much less is known about AII because it is present in far smaller amounts and localized in less accessible deep tissues, primarily kidney. We now report the application of enzymatic and immunologic methods to assess the independent expression and regulation of these two gene products in normal tissue extracts, two cultured cell lines, and multiple organ samples from a hyperargininemic patient who came to autopsy after an unusually severe clinical course characterized by rapidly progressive hepatic cirrhosis. AI was totally absent (less than 0.1%) in the patient's tissues, whereas marked enhancement of AII activity (four times normal) was seen in the kidney by immunoprecipitation and biochemical inhibition studies. Immunoprecipitation-competition and Western blot analysis failed to reveal presence of even an enzymatically inactive cross-reacting AI protein, whereas Southern blot analysis showed no evidence of a substantial deletion in the AI gene. Induction studies in cell lines that similarly express only the AII isozyme indicated that its activity could be enhanced severalfold by exposure to elevated arginine levels. Our findings suggest that the same induction mechanism may well be operative in hyperargininemic patients, and that the heightened AII activity may be responsible for the persistent ureagenesis seen in this disorder. These data lend further support to the existence of two separate arginase gene loci in humans, and raise possibilities for novel therapeutic approaches based on their independent manipulation.

Friedreich's ataxia GAA repeat expansion in patients with recessive or sporadic ataxia.

To explore the clinical heterogeneity associated with the Friedreich's ataxia (FRDA) expanded repeat and provide preliminary guidance for future gene testing in patients suspected of having FRDA, we tested patients with typical FRDA (group I), late-onset FRDA or FRDA with retained reflexes (group II), as well as those with early onset "non-Friedreich's" recessive or sporadic ataxia (group III). Eighty-seven percent of families in group I tested positive for the FRDA triplet repeat expansion. Thirty-six percent of families in group II demonstrated the FRDA expansion. Only one of 11 patients in group III had the FRDA expansion. Clinical criteria did not clearly distinguish between expansion-positive and expansion-negative individuals in groups I and II. Minimal criteria that were present in all the patients who tested positive were recessive or sporadic inheritance, progressive caudal-rostral gait and limb ataxia, and at least one of the following: dysarthria, Babinski sign, or cardiomyopathy. This study confirms recent findings that some patients in group II can carry the FRDA mutation. However, we did not observe the FRDA expansion in 64% of group II families or in 13% of families with typical FRDA (group I), suggesting other genetic or environmental causes for their ataxia.

Minimal or no cancer in radical prostatectomy specimens. Report of 13 cases of the "vanishing cancer phenomenon".

Early detection efforts identify prostate cancer at lower clinical and pathologic stages, often resulting in smaller volumes of tumor in radical prostatectomy specimens. In some cases, complete sampling of the radical prostatectomy specimen for biopsy-proven adenocarcinoma reveals minimal or no residual cancer. We evaluated the clinical and pathologic findings in 13 such cases in an effort to document this finding, which we refer to as the "vanishing cancer phenomenon." The mean number of prostate slides examined per case was 79 (range, 34-248). Carcinoma was absent in two cases, present in a single focus in eight cases, and present in two foci in three cases. Mean cancer volume in the 10 cases with residual tumor was 0.019 cc (range, 0.003-0.038); the largest single dimension of any tumor focus was 3 mm. All cancers were well differentiated or moderately differentiated in the biopsy and prostatectomy. Our results indicate that in some cases cancer may be extremely difficult or impossible to find in the prostatectomy specimen despite exhaustive sampling. The incidence of this "vanishing cancer phenomenon" is probably increasing because more low-stage cancers are being treated by prostatectomy. The inability to identify cancer in a prostate removed for needle biopsy-proven carcinoma may not indicate technical failure.

Gastrointestinal lymphomas. Immunohistochemical studies on the cell of origin.

The classification of primary malignant lymphomas of the gastrointestinal tract by their cell of origin has been a subject of great controversy in recent years, with the proportion of histologic subtypes varying substantially in different published series. Much of this controversy was initially due to the widely recognized inherent difficulty of classifying lymphomas based on routine histologic sections alone. However, the advent of immunohistochemical techniques has also yielded disparate results. Particularly contentious has been the notion of true histiocytic lymphomas, which some investigators have claimed to be relatively frequent in the gastrointestinal tract, whereas others doubt whether they exist at all. We present here a classification of 25 gastrointestinal lymphomas seen in the surgical pathology services of UCLA Hospital and Stanford University Medical Center. Unlike all previously reported series, we have utilized frozen tissue sections for the performance of immunohistochemical studies, which we and others have found to be far more reliable than the use of formalin-fixed, paraffin-embedded tissues, particularly in detecting monoclonal surface staining of immunoglobulin light- and heavy-chain markers. We find that this technique lessens the likelihood of overinterpreting the stains for histiocyte markers (alpha 1-antitrypsin and lysozyme), which are often difficult to read owing to strong positive staining of benign reactive histiocytes within the tumor. Utilizing these techniques, we have been able to classify definitely 21 of our 25 lymphomas (84%) as of B-cell origin, whereas none appeared to be histiocytic. We conclude that true histiocytic lymphomas of the gastrointestinal tract must be very rare, and we recommend the routine use of frozen tissue sections for more accurate classification of these interesting lesions.

Donor origin of a posttransplant liver allograft malignancy identified by fluorescence in situ hybridization for the Y chromosome and DNA genotyping.

Posttransplantation malignancy in the allograft is a rare complication of orthotopic liver transplantation. In the described case, an abnormal T-tube cholangiogram, performed 6 months after orthotopic liver transplantation between a male donor and a female recipient, prompted needle liver biopsy. A moderately differentiated adenocarcinoma was found. Fluorescence in situ hybridization for the Y chromosome indicated male origin of malignancy. Donor-related disease was confirmed by comparative DNA analysis of genomic sequences from the donor liver, associated tumor, and recipient peripheral blood. Results of these investigations qualified the recipient for a second liver transplant.

Infection of the heart by the human immunodeficiency virus.

Heart muscle disease in the acquired immune deficiency syndrome (AIDS), characterized by electrocardiographic changes or congestive cardiomyopathy, is a documented clinical problem, but its pathogenesis is obscure. In AIDS the heart is known to be involved by a variety of opportunistic infections as well as Kaposi's sarcoma, but no causative relation with the development of cardiomyopathy has been established. This study reports evidence for direct infection of the heart in AIDS, not by an opportunistic pathogen but by the AIDS, not by an opportunistic pathogen but by the AIDS virus itself, the human immunodeficiency virus (HIV). For this study the technique of in situ deoxyribonucleic acid hybridization was applied to cardiac tissues obtained at autopsy from AIDS patients. Using sulfur-35-labeled ribonucleic acid probes encompassing the entire HIV genome, HIV nucleic acid sequences were detected in cardiac tissue sections from 6 of 22 patients examined who died of AIDS. The hybridization targets appeared to be cardiac myocytes, although their precise morphology was often obscured by the intensity of the signal. The myocardial cells showing a positive hybridization signal were sparse, often comprising only 1 or a few cells per section, and their number and location did not correlate obviously with any histopathologic or clinical evidence of heart muscle disease in these patients. It is conceivable that the presence of HIV nucleic acid sequences may represent a preclinical marker of impending AIDS-associated heart muscle disease. This sequela would not be recognized in many patients, including those in this series, who died rapidly of Pneumocystis carinii pneumonia, Kaposi's sarcoma and other well-documented manifestations of AIDS.(ABSTRACT TRUNCATED AT 250 WORDS)

Prevalence of the factor V-Leiden mutation in four distinct American ethnic populations.

Resistance to activated protein C (APC) is the most common risk factor for venous thromboembolism, a major cause of morbidity and mortality with an incidence of about 1/1,000 per year. The Arg 506 to Gln mutation in exon 10 of the coagulation factor V gene (factor V-Leiden) has been found to be responsible for over 90% of the APC resistance cases and is an autosomal dominant trait. Initial studies have suggested that this mutation is restricted to individuals of European Caucasian extraction with an average allele frequency in European and American Caucasians of 4.4%, making it one of the most common monogenic disorders in the Caucasian population. A limited number of other ethnic populations have been tested and the mutation has been found only rarely. In our multiethnic survey of 602 individuals, Hispanic-Americans had the highest observed frequency of the factor V-Leiden mutant allele, 1.65%, while African-Americans had a somewhat lower frequency, 0.87%. No factor V-Leiden mutations were found in 191 Asian-Americans or 54 Native-Americans tested. These results indicate that the factor V-Leiden mutation segregates in populations with significant Caucasian admixture and is rare in genetically distant non-European groups. This ethnic stratification may be important in developing cost-effective selective screening programs to identify individuals at risk for thromboembolism and offer prophylactic therapy.

Informativeness of VNTR genetic markers for detecting chimerism after bone marrow transplantation.

We tested the feasibility and practicability of using VNTR-type DNA genetic markers to identify chimerism in the bone marrow of patients following bone marrow transplantation. We selected eight probes which were highly polymorphic when hybridized to DNA digested with MspI. In over 50 donor-recipient combinations, autoradiograms of Southern blots yielded patterns which allowed donor DNA to be distinguished from recipient DNA after an average of two or three serial hybridizations. The strong hybridization signal of VNTR probes also made it possible to detect as little as 1 per cent DNA in DNA mixing experiments.

Detection of cytomegalovirus DNA in classic and epidemic Kaposi's sarcoma by in situ hybridization.

Several lines of evidence have suggested an etiologic association of cytomegalovirus (CMV) with Kaposi's sarcoma. This contention is supported by a pathoepidemiologic survey of 54 cases of acquired immunodeficiency syndrome (AIDS) at our own institution. Of the 27 patients with documented Kaposi's sarcoma, 24 (89%) showed histologic evidence of CMV infection (cytomegalic cells with viral inclusions), whereas only 9 (33%) of the patients with AIDS without Kaposi's sarcoma showed hallmarks of CMV infection. In an attempt to address this question further, we have searched for the presence of CMV nucleic acid sequences in a series of 25 patients with AIDS and Kaposi's sarcoma, using the technique of in situ DNA hybridization. The reliability of the in situ technique is demonstrated, and the technique is shown to be more sensitive than the detection of viral inclusions within Kaposi's sarcoma lesions by routine light microscopy. However, only 20% of our cases showed evidence of CMV involvement, and the CMV-positive cells within the affected Kaposi's sarcoma lesions were few and sparsely distributed. In addition, a companion series of 6 elderly patients with "classic" Kaposi's sarcoma showed no evidence of CMV infection by either conventional microscopy or in situ hybridization. These results do not support the notion of a strong association between Kaposi's sarcoma and CMV, unless the CMV sequences are present at a copy number too low for detection by these methods. The implications of these findings in light of current theories of CMV oncogenesis are discussed.

Immunohistochemical evaluation of K-ras, p53, and HER-2/neu expression in hyperplastic, dysplastic, and carcinomatous lesions of the pancreas: evidence for multistep carcinogenesis.

The pathobiology of precursor lesions leading to invasive pancreatic adenocarcinoma remains a controversial area, but knowledge of the mechanisms of tumorigenesis may lead to possibly earlier detection, prevention, and treatment in the future. We hypothesize that ductal hyperplasia and dysplasia of the pancreas represent precursor lesions and are part of a continuous developmental spectrum evolving into ductal adenocarcinoma of the pancreas. To further define this sequence, we studied the immunohistochemical markers HER-2/neu, K-ras, and p53 in 15 adenocarcinomas and 15 nonmalignant specimens of the pancreas. The 15 nonmalignant specimens of the pancreas included both normal pancreas and chronic pancreatitis. Overall, HER-2/neu was positive in normal ducts, ductal hyperplasia, dysplasia, and carcinoma cells in 0 of 30, 11 of 20 (55%), 10 of 15 (67%), and 12 of 15 (80%), respectively, with progressive increase in the intensity of staining; p53 was positive in 1 of 30 (3%), 0 of 20, 3 of 15 (20%), and 13 of 15 (80%), respectively, and K-ras was positive in 1 of 30 (3%), 6 of 20 (30%), 11 of 15 (73%), and 8 of 15% (53%), respectively. These data support the hypothesis that ductal hyperplasia and dysplasia of the pancreas represent precursor lesions, and, in a fashion similar to that in colorectal tumorigenesis, pancreatic cancer seems to accumulate progressive genetic alterations.

Thymus involution in the acquired immunodeficiency syndrome.

Acquired immunodeficiency syndrome (AIDS) is a severe disorder of unknown etiology and pathogenesis, predominantly affecting homosexual males and other high-risk groups and characterized by profound alterations in T-lymphocyte function. The authors have examined thymus tissue from 14 patients who died of AIDS and compared the results with findings in five control groups: healthy age-matched controls, elderly individuals, patients with chronic or debilitating illnesses other than AIDS, infants with conditions causing "stress atrophy," and patients with myasthenia gravis. The AIDS group included 11 homosexual males, 1 Haitian, 1 homosexual who was also a drug abuser, and a 10-month-old infant believed to have contracted AIDS following blood transfusion. All the AIDS cases showed marked thymus involution with severe depletion of both lymphocytes and epithelial elements. The latter component consisted primarily of thin cords and nests of primitive-appearing epithelial cells that could be defined by positive immunohistochemical staining for keratin. Many cases showed a variable plasma cell infiltration, and the majority exhibited distinct vascular changes in the form of hyalinization and/or onion-skin patterns, primarily in the adventitia. Most striking of all was the marked paucity of Hassall's corpuscles; four patients had none at all, while in the other ten patients all the Hassall's corpuscles were calcified. These changes were far more extensive than those seen in any of the control groups, which retained most of their complement of Hassall's corpuscles even in the face of marked overall involution. The physiologic function of Hassall's corpuscles is not known, but recent immunohistochemical studies have implicated them in the synthesis of "facteur thymique serique" (FTS, thymulin) and other thymic hormones known to play a role in regulating T-helper and suppressor cell activity. It is conceivable that the extensive destruction of Hassall's corpuscles observed in AIDS may be a crucial element in the pathogenesis of this syndrome.

Rapid diagnosis of Legionella infection by a nonisotopic in situ hybridization method.

The authors report a nonradioactive adaptation of DNA hybridization technology for the direct detection of Legionella organisms in situ in routinely processed histologic specimens. The probe used consisted of synthetic oligodeoxynucleotides, complementary to the ribosomal RNA of all clinically relevant Legionella species, labeled with biotinylated dUTP at their 3' ends. By in situ DNA hybridization and detection with an avidin-alkaline phosphatase complex. Legionella was visualized by light microscopy within the alveoli of lung specimens in 9 of 13 direct fluorescent antibody- or culture-positive cases of Legionnaires' disease. No cross-hybridization was observed in lung specimens infected with Pseudomonas aeruginosa, Klebsiella pneumoniae, Streptococcus pneumoniae, or other pathogens. The authors' results illustrate a novel adaptation of in situ DNA hybridization techniques, usually used for viruses, to the detection of a bacterial organism. The method enables direct visualization of bacterial nucleic acid in infected tissues and may facilitate early diagnosis and treatment of legionellosis.

Polymerase chain reaction-based K-ras mutation detection of pancreatic adenocarcinoma in routine cytology smears.

The cytologic diagnosis of pancreatic carcinoma is notoriously difficult, particularly in distinguishing benign atypia from well-differentiated adenocarcinoma. Mutation of codon 12 in the K-ras oncogene is frequently found with pancreatic cancers. Detection by polymerase chain reaction (PCR) followed by restriction endonuclease digestion can provide a powerful tool to improve and confirm diagnosis. The authors examined the utility of PCR-based detection in the diagnosis of pancreatic carcinoma using routinely obtained cytology smears that could be collected at most hospitals. Pancreatic cytology smears were collected retrospectively from 60 patients. DNA was extracted from the slides and amplified by PCR using mismatched primers that generated a Bst-N1 recognition site with the wild type codon 12 but not with the mutant allele. Results were compared with clinical follow-up. K-ras codon 12 mutations were observed in 44 of 46 (95.7%) cases of pancreatic cancer, but not in 12 benign cases nor in 2 cases of islet cell tumor. The amplification and digestion steps proved robust and sensitive, capable of detecting mutant K-ras alleles from cytology smears that contained only small foci of suspicious cells. Our results indicate that K-ras mutation analysis can be done reliably within 1 to 2 days from routine cytology slides without special handling, increasing the sensitivity of diagnosis in ambiguous cases while maintaining cost-effective and relatively noninvasive sampling strategy.

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.