A multicenter analysis of individuals with a 47,XXY/46,XX karyotype.

PURPOSE: Klinefelter syndrome, a sex chromosome aneuploidy (SCA), is associated with a 47,XXY chromosomal complement and is diagnosed in ∼1:600 live male births. Individuals with a 46,XX cell line, in addition to 47,XXY, are less common with a limited number of published case reports. METHODOLOGY: To better understand the implications of a 47,XXY/46,XX karyotype, we conducted a retrospective, multicenter analysis of the cytogenetic findings and associated clinical records of 34 patients diagnosed with this SCA across 14 institutions. RESULTS: Presence of the XX cell line ranged from 5% to 98% in patient specimens. Phenotypes also exhibited significant heterogeneity with some reporting a single reason for referral and others presenting with a constellation of symptoms, including ambiguous genitalia and ovotestes. Ovotestes were present in 12% of individuals in this cohort, who had a significantly higher percentage of XX cells. Notably, 2 patients were assigned female sex at birth. CONCLUSION: These findings highlight the variability of the clinical phenotypes associated with this SCA, as well as the challenges of clinical management for this population. Karyotype or fluorescence in situ hybridization analysis, which offer single-cell resolution, rather than chromosomal microarray or molecular testing, is the ideal test strategy in these instances as mosaicism can occur at low levels.

Clinical diagnosis of neurofibromatosis type I in multiple family members due to cosegregation of a unique balanced translocation with disruption of the NF1 locus: Testing considerations for accurate diagnosis.

Neurofibromatosis type 1 (NF1) is an autosomal dominant disorder that causes a predisposition to develop tumors along the peripheral nervous system. The NF1 gene, located at 17q11.2, has the highest mutation rate among known human genes and about half of NF1 patients have de novo pathogenic variants. We present a case of clinical NF1 diagnoses in multiple family members with phenotypes ranging from mild to severe. Chromosome analysis of the 3-year-old female proband with NF1 resulted in an abnormal karyotype that was inherited from her mother: 46,XX,t(4;17)(q21.3;q11.2) mat. However, no NF1 genetic variants were identified by either NGS analysis of NF1 DNA coding regions, deletion-duplication studies, or by cytogenomic microarray copy number analysis. Follow-up chromosome studies of the proband's two male siblings demonstrated cosegregation of the same balanced translocation and a clinical diagnosis of NF1. Based on the cosegregation of the translocation with the NF1 clinical presentation in this family, we hypothesized that the NF1 gene may have been disrupted by this unique rearrangement. Subsequent fluorescence in situ hybridization (FISH) analysis of the metaphase cells of an affected sibling revealed a disruption of the NF1 gene confirming the underlying basis of the clinical NF1 presentation in this family. The utilization of traditional cytogenetic as well as evolving molecular methods was not only pivotal in the diagnosis of NF1 and management for this family, but is also pertinent to other patients with a family history of NF1.

Bone Marrow-derived Cells Contribute to the Pathogenesis of Pulmonary Arterial Hypertension.

RATIONALE: Pulmonary arterial hypertension (PAH) is a progressive lung disease of the pulmonary microvasculature. Studies suggest that bone marrow (BM)-derived circulating cells may play an important role in its pathogenesis. OBJECTIVES: We used a genetic model of PAH, the Bmpr2 mutant mouse, to study the role of BM-derived circulating cells in its pathogenesis. METHODS: Recipient mice, either Bmpr2(R899X) mutant or controls, were lethally irradiated and transplanted with either control or Bmpr2(R899X) BM cells. Donor cells were traced in female recipient mice by Y chromosome painting. Molecular and function insights were provided by expression and cytokine arrays combined with flow cytometry, colony-forming assays, and competitive transplant assays. MEASUREMENTS AND MAIN RESULTS: We found that mutant BM cells caused PAH with remodeling and inflammation when transplanted into control mice, whereas control BM cells had a protective effect against the development of disease, when transplanted into mutant mice. Donor BM-derived cells were present in the lungs of recipient mice. Functional and molecular analysis identified mutant BM cell dysfunction suggestive of a PAH phenotype soon after activation of the transgene and long before the development of lung pathology. CONCLUSIONS: Our data show that BM cells played a key role in PAH pathogenesis and that the transplanted BM cells were able to drive the lung phenotype in a myeloablative transplant model. Furthermore, the specific cell types involved were derived from hematopoietic stem cells and exhibit dysfunction long before the development of lung pathology.

The duplication 17p13.3 phenotype: analysis of 21 families delineates developmental, behavioral and brain abnormalities, and rare variant phenotypes.

Chromosome 17p13.3 is a gene rich region that when deleted is associated with the well-known Miller-Dieker syndrome. A recently described duplication syndrome involving this region has been associated with intellectual impairment, autism and occasional brain MRI abnormalities. We report 34 additional patients from 21 families to further delineate the clinical, neurological, behavioral, and brain imaging findings. We found a highly diverse phenotype with inter- and intrafamilial variability, especially in cognitive development. The most specific phenotype occurred in individuals with large duplications that include both the YWHAE and LIS1 genes. These patients had a relatively distinct facial phenotype and frequent structural brain abnormalities involving the corpus callosum, cerebellar vermis, and cranial base. Autism spectrum disorders were seen in a third of duplication probands, most commonly in those with duplications of YWHAE and flanking genes such as CRK. The typical neurobehavioral phenotype was usually seen in those with the larger duplications. We did not confirm the association of early overgrowth with involvement of YWHAE and CRK, or growth failure with duplications of LIS1. Older patients were often overweight. Three variant phenotypes included cleft lip/palate (CLP), split hand/foot with long bone deficiency (SHFLD), and a connective tissue phenotype resembling Marfan syndrome. The duplications in patients with clefts appear to disrupt ABR, while the SHFLD phenotype was associated with duplication of BHLHA9 as noted in two recent reports. The connective tissue phenotype did not have a convincing critical region. Our experience with this large cohort expands knowledge of this diverse duplication syndrome.

Collaborative efforts to improve genetic testing in the neonatal intensive care unit.

OBJECTIVE: To reduce unnecessary simultaneous karyotype analysis and chromosomal microarray (CMA) testing in the neonatal intensive care unit (NICU). STUDY DESIGN: This quality improvement study investigated the effect of collaborative efforts between the NICU, cytogenetics, and clinical genetics on numbers of genetic tests, rates of abnormal tests, and number of genetics consults comparing baseline and 5-month intervention periods. RESULTS: Simultaneous karyotype analyses and CMAs decreased due to a decrease in karyotype testing (11.3% [68/600] vs. 0.98% [6/614], p < 0.01). Karyotype analyses were more likely to be abnormal (13.8% [12/87] vs. 64.0% [16/25], p < 0.01). Frequency of genetics consultation did not change (7.0% [42/600] vs. 9.4% [58/614], p = 0.12). CONCLUSION: Collaborative efforts between the NICU, cytogenetics, and clinical genetics decreased redundant genetic testing, which demonstrated potential cost savings to our institution. Ongoing collaborative efforts could facilitate genetic testing practices in the NICU that readily evolve in tandem with genetic testing recommendations.

Tnni3k modifies disease progression in murine models of cardiomyopathy.

The Calsequestrin (Csq) transgenic mouse model of cardiomyopathy exhibits wide variation in phenotypic progression dependent on genetic background. Seven heart failure modifier (Hrtfm) loci modify disease progression and outcome. Here we report Tnni3k (cardiac Troponin I-interacting kinase) as the gene underlying Hrtfm2. Strains with the more susceptible phenotype exhibit high transcript levels while less susceptible strains show dramatically reduced transcript levels. This decrease is caused by an intronic SNP in low-transcript strains that activates a cryptic splice site leading to a frameshifted transcript, followed by nonsense-mediated decay of message and an absence of detectable protein. A transgenic animal overexpressing human TNNI3K alone exhibits no cardiac phenotype. However, TNNI3K/Csq double transgenics display severely impaired systolic function and reduced survival, indicating that TNNI3K expression modifies disease progression. TNNI3K expression also accelerates disease progression in a pressure-overload model of heart failure. These combined data demonstrate that Tnni3k plays a critical role in the modulation of different forms of heart disease, and this protein may provide a novel target for therapeutic intervention.

Distinct Patterns of Clonal Evolution Drive Myelodysplastic Syndrome Progression to Secondary Acute Myeloid Leukemia.

Clonal evolution in myelodysplastic syndrome (MDS) can result in clinical progression and secondary acute myeloid leukemia (sAML). To dissect changes in clonal architecture associated with this progression, we performed single-cell genotyping of paired MDS and sAML samples from 18 patients. Analysis of single-cell genotypes revealed patient-specific clonal evolution and enabled the assessment of single-cell mutational cooccurrence. We discovered that changes in clonal architecture proceed via distinct patterns, classified as static or dynamic, with dynamic clonal architectures having a more proliferative phenotype by blast count fold change. Proteogenomic analysis of a subset of patients confirmed that pathogenic mutations were primarily confined to primitive and mature myeloid cells, though we also identify rare but present mutations in lymphocyte subsets. Single-cell transcriptomic analysis of paired sample sets further identified gene sets and signaling pathways involved in two cases of progression. Together, these data define serial changes in the MDS clonal landscape with clinical and therapeutic implications. SIGNIFICANCE: Precise clonal trajectories in MDS progression are made possible by single-cell genomic sequencing. Here we use this technology to uncover the patterns of clonal architecture and clonal evolution that drive the transformation to secondary AML. We further define the phenotypic and transcriptional changes of disease progression at the single-cell level. See related article by Menssen et al., p. 330 (31). See related commentary by Romine and van Galen, p. 270. This article is highlighted in the In This Issue feature, p. 265.

Acquisition of aneuploidy drives mutant p53-associated gain-of-function phenotypes.

p53 is mutated in over half of human cancers. In addition to losing wild-type (WT) tumor-suppressive function, mutant p53 proteins are proposed to acquire gain-of-function (GOF) activity, leading to novel oncogenic phenotypes. To study mutant p53 GOF mechanisms and phenotypes, we genetically engineered non-transformed and tumor-derived WT p53 cell line models to express endogenous missense mutant p53 (R175H and R273H) or to be deficient for p53 protein (null). Characterization of the models, which initially differed only by TP53 genotype, revealed that aneuploidy frequently occurred in mutant p53-expressing cells. GOF phenotypes occurred clonally in vitro and in vivo, were independent of p53 alteration and correlated with increased aneuploidy. Further, analysis of outcome data revealed that individuals with aneuploid-high tumors displayed unfavorable prognoses, regardless of the TP53 genotype. Our results indicate that genetic variation resulting from aneuploidy accounts for the diversity of previously reported mutant p53 GOF phenotypes.

Institutional Training Opportunities for PhD Students in Laboratory Medicine: An Unmet Career Development Need?

In the United States, the credentialing of PhD-scientists as medical directors of clinical laboratories is driven by formal postdoctoral training programs. Prior to acceptance in one these accredited fellowships, however, a trainee's exposure to the field can be far less standardized, with significant ramifications for their awareness and competitiveness. In the current article, we describe our recent experiences in developing local, institution-based immersion opportunities for PhD experiences in the subdisciplines of laboratory medicine (clinical microbiology, clinical chemistry, and molecular genetics/genomics). It is our hope that this article-and a corresponding online survey-can prompt reflection and discussion on the status of early career training opportunities in these key clinical areas.

A quantitative trait locus (LSq-1) on mouse chromosome 7 is linked to the absence of tissue loss after surgical hindlimb ischemia.

BACKGROUND: Peripheral arterial disease (PAD) caused by occlusive atherosclerosis of the lower extremity has 2 major clinical manifestations. Critical limb ischemia is characterized by rest pain and/or tissue loss and has a > or = 40% risk of death and major amputation. Intermittent claudication causes pain on walking, has no tissue loss, and has amputation plus mortality rates of 2% to 4% per year. Progression from claudication to limb ischemia is infrequent. Risk factors in most PAD patients overlap. Thus, we hypothesized that genetic variations may be linked to presence or absence of tissue loss in PAD. METHODS AND RESULTS: Hindlimb ischemia (murine model of PAD) was induced in C57BL/6, BALB/c, C57BL/6 x BALB/c (F1), F1 x BALB/c (N2), A/J, and C57BL/6J-Chr7(A/J)/NaJ chromosome substitution strains. Mice were monitored for perfusion recovery and tissue necrosis. Genome-wide scanning with polymorphic markers across the 19 murine autosomes was performed on the N2 mice. Greater tissue loss and poorer perfusion recovery occurred in BALB/c than in the C57BL/6 strain. Analysis of 105 N2 progeny identified a single quantitative trait locus on chromosome 7 that exhibited significant linkage to both tissue necrosis and extent of perfusion recovery. Using the appropriate chromosome substitution strain, we demonstrate that C57BL/6-derived chromosome 7 is required for tissue preservation. CONCLUSIONS: We have identified a quantitative trait locus on murine chromosome 7 (LSq-1) that is associated with the absence of tissue loss in a preclinical model of PAD and may be useful in identifying gene(s) that influence PAD in humans.

Preserved expressive language as a phenotypic determinant of Mosaic Angelman Syndrome.

BACKGROUND: Angelman Syndrome (AS) is a neurodevelopmental disorder with core features of intellectual disability, speech impairment, movement disorders, and a unique behavioral profile. Typically, AS results from absent maternal expression of UBE3A, but some individuals have imprinting defects in a portion of their cells. These individuals are mosaic for normal and defective UBE3A expression, resulting in mosaic AS (mAS) with a partial loss of gene expression. METHODS: This study aims to contrast the mAS phenotype to that of AS. Clinical characteristics of mAS were obtained from a parental survey of 22 mAS patients and from the Angelman Natural History study. These were contrasted with those of AS using historical data. RESULTS: Developmental delay was present in nearly all mAS patients, whereas the core features of AS were reported in less than 40%. While language and ability to manage activities of daily living were markedly improved over that expected in AS, mAS patients demonstrated a high incidence of behavioral challenges. CONCLUSION: Clinical work-up of an individual with developmental delay, hyperactivity, anxiety, and an uncharacteristically happy demeanor should prompt methylation studies to rule out mAS. We expand the phenotypic spectrum of AS to include features that overlap with Prader-Willi such as hyperphagia.

A unique evolution of the kidney phenotype in a patient with autosomal recessive Alport syndrome.

Alport syndrome is due to mutations in one of the genes encoding (α3,4,5) type IV collagen resulting in defective type IV collagen, a key component of the glomerular basement membrane (GBM). The GBM is initially thin and, with ongoing remodeling, develops a thickened basket-woven appearance. We report a unique case of a 9-year-old boy who underwent biopsy for hematuria and proteinuria, diagnosed as IgA nephropathy, with normal GBM appearance and thickness. Because of a family history of hematuria and chronic kidney disease, he subsequently underwent genetic evaluation, and a mutation of α3 type IV collagen (COL4A3) was detected. Additional studies of the initial biopsy demonstrated abnormal type IV collagen immunostaining. A repeat biopsy 4 years later showed characteristic glomerular basement membrane morphology of Alport syndrome and scarring consistent with sequelae of IgA nephropathy. This is the first description of this unusual transition from an initial normal appearance of the glomerular basement membrane to the classic Alport phenotype.

Limited Utility of Fluorescence In Situ Hybridization for Recurrent Abnormalities in Acute Myeloid Leukemia at Diagnosis and Follow-up.

OBJECTIVES: Acute myeloid leukemia (AML) is classified in part by recurrent cytogenetic abnormalities, often detected by both fluorescent in situ hybridization (FISH) and karyotype. The goal of this study was to assess the utility of FISH and karyotyping at diagnosis and follow-up. METHODS: Adult AML samples at diagnosis or follow-up with karyotype and FISH were identified. Concordance was determined, and clinical characteristics and outcomes for discordant results were evaluated. RESULTS: Karyotype and FISH results were concordant in 193 (95.0%) of 203 diagnostic samples. In 10 cases, FISH detected an abnormality, but karyotype was normal. Of these, one had a FISH result with clinical significance. In follow-up cases, 17 (8.1%) of 211 showed FISH-positive discordant results; most were consistent with low-level residual disease. CONCLUSIONS: Clinically significant discordance between karyotype and AML FISH is uncommon. Consequently, FISH testing can safely be omitted from most of these samples. Focused FISH testing is more useful at follow-up, for minimal residual disease detection.

Association of hereditary prostate cancer gene polymorphic variants with sporadic aggressive prostate carcinoma.

BACKGROUND: ELAC2, MSR1, and RNASEL are candidate genes for hereditary prostate carcinoma (HPC). While, studies have demonstrated that single nucleotide polymorphisms (SNPs) in these genes are associated with sporadic disease as well as HPC, these results are often not replicated in follow-up studies. Given that the majority of patients studied had localized disease and up to 50% of localized prostate cancer is clinically insignificant, the inability to replicate the initial findings may reflect that some subjects had indolent tumors. Herein, we examine patients with metastatic disease to determine if an association exists between HPC SNPs and unambiguously significant prostate cancer. METHODS: We examined polymorphisms within ELAC2 (S217L, A541T, E622V), MSR1 (P275A, R293X, aIVS5-59c), and RNASEL (E265X, R462Q, D541E) in 150 European-Americans with metastatic prostate cancer and 170 prostate cancer-free controls using pyrosequencing assays. RESULTS: Only ELAC2 217L (37% cases vs. 29% controls (P=0.034)) and RNASEL 541E (61% cases vs. 53% controls (P=0.045)) were over-represented. Analysis of genotypes revealed that presence of the leucine ELAC2 allele (OR 1.54: 95% CI=0.99-2.41, SS vs. SL, LL) and homozygosity for the glutamic acid RNASEL allele (OR 1.68: 95% CI=1.04-2.70, EE vs. DE, DD) were associated with increased risk. Patients with both genotypes were of particularly high-risk (OR 2.66: 95% CI=1.36-5.19). CONCLUSIONS: These results suggest that, in a European-American population, ELAC2 217L and RNASEL 541E are associated with metastatic sporadic disease. ELAC2 and RNASEL SNP analysis may prove useful in determining which patients are at risk for developing clinically significant prostate carcinoma.

QTL mapping in a mouse model of cardiomyopathy reveals an ancestral modifier allele affecting heart function and survival.

The progression from myocardial hypertrophy to heart failure is a complex process, involving genetic and environmental factors. Elucidating the genetic components contributing to heart failure has been difficult, largely because of the heterogeneity of human populations. We have employed a strategy to map genetic loci that modify the heart failure phenotype in a transgenic mouse model of cardiomyopathy caused by cardiac-specific overexpression of calsequestrin. Strain-specific differences in both cardiac function and survival are observed when the transgene is moved into different inbred mouse strains. We have previously reported linkage results from mapping in reciprocal backcrosses between C57/BL6 (BL6) and DBA/2J (DBA) and a backcross between DBA/AKR and AKR. Here we report the results of a genome-wide linkage scan in the reciprocal backcross between DBA/AKR and DBA. We identified one novel locus on Chromosome (Chr) 18 that affects heart function and a second on Chr 3 that shows significant linkage to both survival and heart function. Intriguingly, the Chr 3 allele of AKR shows a susceptibility effect on phenotype, whereas the overall effect of the AKR genetic background is protective. The Chr 3 locus also completely overlaps the Hrtfm2 locus, which was previously mapped in crosses between DBA and BL6. Mapping the same QTL in two different crosses allowed us to use ancestral haplotypes to narrow the candidate gene interval from 9 to 2 Mb. Identification of the genes at these QTLs in the mouse will provide novel candidate genes that can be evaluated for their role in human heart failure.