New treatments for serious conditions: ethical implications.

Approval of Spinraza (nusinersen) for treatment of spinal muscular atrophy prompts consideration of a number of ethical issues that arise whenever a new treatment is proposed for a serious condition, especially one that is rare and can devastatingly affect children. Patients, families, clinicians, researchers, institutions and policymakers all must take account of the ways that newly available treatments affect informed and shared decision-making about therapeutic and research options. The issues to consider include: addressing what is still uncertain and unknown; the possibility that potential benefits will be exaggerated and potential harms underemphasized in the media, by advocacy organizations, and in consent forms and processes; the high cost of many novel drugs and biologics; the effects of including conditions of variable phenotype in state-mandated newborn screening panels; and how new treatments can change the standard of care, altering what is and is not known about a disorder and posing challenges for decision-making at both individual and policy levels. The good news that Spinraza brings thus requires additional attention to its ethical and policy implications, to improve counseling and shared decision-making about treatment and research options for patients and all involved in their care.

Embryoid body formation of human amniotic fluid stem cells depends on mTOR.

Human amniotic fluid stem cells (hAFSCs) harbor high proliferative capacity and high differentiation potential and do not raise the ethical concerns associated with human embryonic stem cells. The formation of three-dimensional aggregates known as embryoid bodies (EBs) is the principal step in the differentiation of pluripotent embryonic stem cells. Using c-Kit-positive hAFSC lines, we show here that these stem cells harbor the potential to form EBs. As part of the two kinase complexes, mTORC1 and mTORC2, mammalian target of rapamycin (mTOR) is the key component of an important signaling pathway, which is involved in the regulation of cell proliferation, growth, tumor development and differentiation. Blocking intracellular mTOR activity through the inhibitor rapamycin or through specific small interfering RNA approaches revealed hAFSC EB formation to depend on mTORC1 and mTORC2. These findings demonstrate hAFSCs to be a new and powerful biological system to recapitulate the three-dimensional and tissue level contexts of in vivo development and identify the mTOR pathway to be essential for this process.

Identification and sequencing the juvenile spermatogonial depletion critical interval on mouse chromosome 1 reveals the presence of eight candidate genes.

In mice, the recessive, non-pleiotropic, juvenile spermatogonial depletion (jsd) mutation results in a single wave of spermatogenesis, followed by failure of type A spermatogonial stem cells to differentiate, rendering adult males sterile. As part of an effort to identify the gene underlying this mutation, we report here the construction of a high-resolution genetic map involving more than 1000 meioses and 24 polymorphic loci. Our data define a critical jsd interval of approximately 0.4 cM at 49 cM on mouse chromosome 1, between D1Mit215 and 257SP6. We have constructed a physical map spanning the region comprising 24 overlapping BACs. Eighteen of these BACs have been fully sequenced, or are in draft form, allowing us to annotate approximately 2.5 Mb of DNA surrounding the jsd locus. The critical 0.4 cM jsd interval corresponds to a physical distance of approximately 1.5 Mb. Eight genes have been identified in this interval, two of which appear to be possible candidates for the jsd mutation.

A transgenic insertion causing cryptorchidism in mice.

A distinctive feature of gonadal maturation in mammals is the movement to an extraabdominal location. Testicular descent is a complex, multistage process whereby the embryonic gonads migrate from their initial abdominal position to the scrotum. Failure in this process results in cryptorchidism, a frequent congenital birth defect in humans. We report here a new mouse transgenic insertional mutation, cryptorchidism with white spotting (crsp). Males homozygous for crsp exhibit a high intraabdominal position of the testes, associated with complete sterility. Heterozygous males have a wild-type phenotype, and homozygous females are fertile. Surgically descended testes in crsp/crsp males show normal spermatogenesis. Using FISH and genetic analyses, the transgenic insert causing the crsp mutation has been mapped to the distal part of mouse chromosome 5. Transgene integration resulted in a 550-kb deletion located upstream of the Brca2 gene. A candidate gene encoding a novel G protein-coupled receptor (Great) with an expression pattern suggesting involvement in testicular descent has been identified.

Smcy transgene does not rescue spermatogenesis in sex-reversed mice.

In mouse, the Sxr(b) deletion interval (delta Sxr(b)) maps to the small short arm of the Y chromosome and is known to contain gene(s) required for normal spermatogenesis; in particular, Spy, which is essential for the postnatal mitotic proliferation of spermatogonia. This deletion interval is approximately 1-2 Mb and contains eight known genes. In this paper we report the construction of YAC transgenic mice containing different regions of the delta Sxr(b) interval including Zfy1, Ube1y, Smcy, and Eif2s3. Two male and one female founder mice, transgenic for all four genes, were sterile. However, a fertile transgenic, carrying a full-length copy of the Smcy gene integrated into central Chr 12, was identified. Smcy is a highly conserved Y chromosome-located gene, encoding peptides corresponding to epitopes of the male-specific antigen, H-Y. The Smcy transgene was ubiquitously expressed in all organs and tissues tested in male and female carriers. Introduction of the transgene into an X Sxr(b)/O genetic background did not rescue the early arrest of spermatogenesis characteristic of these males. These data indicate that the presence of Smcy is not sufficient to restore spermatogenesis, making it a highly unlikely candidate for Spy.

A transgenic insertion upstream of sox9 is associated with dominant XX sex reversal in the mouse.

In most mammals, male development is triggered by the transient expression of the Y-chromosome gene, Sry, which initiates a cascade of gene interactions ultimately leading to the formation of a testis from the indifferent fetal gonad. Several genes, in particular Sox9, have a crucial role in this pathway. Despite this, the direct downstream targets of Sry and the nature of the pathway itself remain to be clearly established. We report here a new dominant insertional mutation, Odsex (Ods), in which XX mice carrying a 150-kb deletion (approximately 1 Mb upstream of Sox9) develop as sterile XX males lacking Sry. During embryogenesis, wild-type XX fetal gonads downregulate Sox9 expression, whereas XY and XX Ods/+ fetal gonads upregulate and maintain its expression. We propose that Ods has removed a long-range, gonad-specific regulatory element that mediates the repression of Sox9 expression in XX fetal gonads. This repression would normally be antagonized by Sry protein in XY embryos. Our data are consistent with Sox9 being a direct downstream target of Sry and provide genetic evidence to support a general repressor model of sex determination in mammals.

Juvenile spermatogonial depletion (jsd) mutant seminiferous tubules are capable of supporting transplanted spermatogenesis.

In mice, the juvenile spermatogonial depletion (jsd) mutation results in a single wave of spermatogenesis followed by failure of type A spermatogonial stem cells to repopulate the testis, rendering male animals sterile. It is not clear whether the defect in jsd resides in a failure of the somatic component to support spermatogenesis or in a failure that is intrinsic to the mutant's germ cells. To determine if the jsd intratesticular environment is capable of supporting spermatogenesis, germ cell transplantation experiments were performed in which C57BL/6 ROSA germ cells were transplanted into jsd recipients. To determine if jsd spermatogonia are able to develop in a permissive seminiferous environment, jsd germ cells were transplanted into W/W(v) and busulfan-treated C57BL/6 animals. The data demonstrate that up to 7 mo after transplantation of normal germ cells, jsd seminiferous tubules are capable of supporting spermatogenesis. In contrast, when jsd germ cells were transplanted into busulfan-treated C57BL/6 testis, or into testis of W/W(v) mice, no jsd-derived spermatogenesis was observed. The data support the hypothesis that the jsd phenotype is due to a defect in the germ cells themselves, and not in the intratubular environment.

Mouse strain susceptibility to diethylnitrosamine induced hepatocarcinogenesis is cell autonomous whereas sex-susceptibility Is due to the micro-environment: analysis with C3H <--> BALB / c sexually chimeric mice.

In man, liver cancer is on the increase, especially in males. Sex differences also exist in rodent models. To elucidate the mechanisms, chimeric mice were produced by amalgamation of early embryos from high and low hepatocarcinogen-susceptible strains, C3H and BALB / c. Tumor formation was initiated with 10 mg / kg of diethylnitrosamine at the ages of 7 and 14 days and mice were sacrificed at 30 and 45 weeks. The chimeras were classified into XY <--> XY, XY <--> XX, XX <--> XY, and XX <--> XX in terms of sex chromosomes by means of polymerase chain reaction-simple sequence length polymorphism analysis (SSLP) using Y chromosome-specific Sry primers in combination with the D3Mit21 marker. Liver lesions were analyzed histopathologically, by immunostaining using a C3H strain-specific antibody and by DNA in situ hybridization with the Y chromosome-specific digoxigenin-labeled Y353 / B probe. Sex and strain genotyping by SSLP analysis matched histological observations, confirming the reliability of our system. The strain differences in liver tumor numbers of each strain type in XY <--> XY and XX <--> XX subtypes of C3H <--> BALB / c chimeras were retained well (P < 0. 0001 and P < 0.001, respectively), indicating a minimum influence of the C3H or BALB / c surrounding milieu on development of individual lesions. On the other hand, significant promotion of XX cell tumors was evident in phenotypically male sexually chimeric XY <--> XX and XX <--> XY chimeras for both C3H (P < 0.02) and BALB / c (P < 0.01) lesions compared to the XX <--> XX case. The results suggest the presence of hormonal or micro-environmental factors specific for males, which are not caused cell-autonomously. Basic strain differences, however, are determined by intrinsic genetic factors rather than the strain-dependent micro-environment.

Efficiency of home care: notes for an economic approach to resource allocation.

Home-delivered health-related services are not valued for themselves but are, rather, highly customized inputs that can be used in the production of several alternative "commodities" ultimately demanded by consumers. These valued commodities are the restoration, improvement, or maintenance of health and functional status that can be produced by home-delivered care (and by alternative services) and the everyday support of daily functioning at home. If we can describe how inputs into the production of these two commodities are allocated, it may be possible to make judgments about whether home-delivered care and other services would produce more value if they were allocated differently.

Where are the missing elders? The decline in nursing home use, 1985 and 1995.

Findings from the 1995 National Nursing Home Survey suggest that elderly Americans are reducing their use of nursing home care. The numbers reflect a change in the role of the nursing home, as defined in this survey. By 1995 nursing facilities were increasingly focusing on patients with greater disability and postacute care needs. Preferred alternatives, most notably home-delivered care and assisted living, were likely filling the gap left by declining nursing home use. Better population-based studies are needed to track emerging trends and ascertain whether elders with disabilities are receiving the care they need. Such data could inform development of better public and private financing strategies for long-term care.

The Medicare home health benefit implications of recent payment changes.

The interim payment system (IPS) for Medicare home health services, enacted in the Balanced Budget Act of 1997, was intended to slow the growth of home health expenses until HCFA could design a new prospective system. Instead, the IPS has acted like a per-case payment system without case-mix adjustment. Its impact on agencies, along with other policy pressures, has been first to slow and then to reverse the dramatic expansion of the home health sector. In this paper, we identify the impetus for payment changes in the recent history of the Medicare home health benefit. We then present emerging evidence about the effects of IPS and other recent policies on home health. Finally, we draw several lessons from this experience for the impending prospective payment system.

Health cost containment: what it will mean for workers and local economies.

After decades of rapid growth, the rate of increase in health services spending appears to be moderating. Although a slowdown in health expenditure growth would release resources for other uses in the economy, concerns have been raised about the effects of a spending slowdown on health workers and regional economies. Based on projections carried out by the Bureau of Labor Statistics during the health reform debate and on state health sector employment data, the author concludes that health workers may experience costly dislocation as health spending growth slows, and some regions may be more affected than others. However, the appropriate response is a general economic policy supporting economic growth and full employment policy with regard to health expenditure growth cannot be held hostage to concerns about employment effects.

Evolution of the DAZ gene family suggests that Y-linked DAZ plays little, or a limited, role in spermatogenesis but underlines a recent African origin for human populations.

The recent transposition to the Y chromosome of the autosomal DAZL1 gene, potentially involved in germ cell development, created a unique opportunity to study the rate of Y chromosome evolution and assess the selective forces that may act upon such genes, and provided a new estimate of the male-to-female mutation rate (alpham). Two different Y-located DAZ sequences were observed in all Old World monkeys, apes and humans. Different DAZ copies originate from independent amplification events in each primate lineage. A comparison of autosomal DAZL1 and Y-linked DAZ intron sequences gave a new figure for male-to-female mutation rates of alpham = 4. It was found that human DAZ exons and introns are evolving at the same rate, implying neutral genetic drift and the absence of any functional selective pressures. We therefore hypothesize that Y-linked DAZ plays little, or a limited, role in human spermatogenesis. The two copies of DAZ in man appear to be due to a relatively recent duplication event (55 000-200 000 years). A worldwide survey of 67 men from five continents representing 19 distinct populations showed that most males have both DAZ variants. This implies a common origin for the Y chromosome consistent with a recent 'out of Africa' origin of the human race.

Transposition of RhoA to the murine Y chromosome.

In an effort to produce a more complete transcription map of the short (approximately 5 Mb) arm of the mouse Y chromosome, we have initiated exon trapping from Yp-derived YACs. Sequence analysis of the trapped products has identified exons of previously cloned mouse Y-located genes Zfy and SSty and potential exons homologous to the human Y-located Tspy gene family. In addition, a family of three Yp-located transcripts that show close homology to human RHOA (locus designation ARHA), a member of the Ras family of small GTPases, has been identified. To determine whether these Yp sequences had been transposed from an autosomal ancestor, we used this trapped product to isolate a full-length autosomal mouse RhoA cDNA that is 80% identical at the nucleotide level and 98% identical at the amino acid level to human RHOA and maps to mouse Chromosome 2 (locus designation ArhA). Sequence analysis indicates that the Y-linked copies have diverged from the autosomal form, with small deletions precluding maintenance of a significant open reading frame in all Yp copies. Yet RT-PCR analysis indicates that two of these pseudogenes, RhoAy1 and 3, are expressed in a testis-specific manner, in sharp contrast to the nearly ubiquitous expression pattern of the autosomal ancestor. The data indicate that the Y copies of RhoA have been transposed from an autosome, followed by subsequent duplication, sequence divergence, and acquisition of a testis-specific promoter/enhancer.