Michael V. Zaragoza

Human aneuploidy: Incidence, origin, and etiology

Chromosome abnormalities occur with astonishing frequency in humans, being present in an estimated 1030% of all fertilized eggs. Of the different classes of chromosome abnormality, aneuploidy (trisomy and monosomy) is by far the most common and, clinically, the most important. For example, over 25% of all miscarriages are monosomic or trisomic, making aneuploidy the leading known cause of pregnancy loss. Furthermore, among those conceptions that survive to term, aneuploidy is the leading genetic cause of mental retardation. Over the past 25 years, a considerable body of information has accrued on the incidence of aneuploidy in human gametes, fetuses, and newborns. More recently, the application of molecular biological techniques to the study of aneuploidy has begun to uncover some of the underlying causes of human aneuploidy. In this review, we first summarize the cytogenetic data on the incidence of aneuploidy in humans, and then discuss recent molecular data on the mechanism of origin of different aneuploid conditions, the basis of the maternal age effect on aneuploidy, and the importance of aberrant genetic recombination to the genesis of aneuploidy.

Nondisjunction of human acrocentric chromosomes : studies of 432 trisomic fetuses and liveborns

The present report summarizes molecular studies on the parent and meiotic stage of origin of the additional chromosome in 432 fetuses or liveborns with an additional chromosome 13, 14, 15, 21, or 22. Our studies suggest that there is little variation in the origin of nondisjunction among the five acrocentric trisomies and that there is no association between the origin of nondisjunction and the likelihood of survival to term of the trisomic conceptus. The proportion of cases of paternal origin was similar among the five trisomies: 12% for trisomy 13, 17% for trisomy 14, 12% for trisomy 15, 9% for trisomy 21, and 11% for trisomy 22. The stage of nondisjunction was also similar among the five trisomies, with the majority of cases of maternal origin being due to nondisjunction at meiosis I, whereas for paternally derived cases, nondisjuction occurred primarily at meiosis II.

Mitochondrial DNA variant discovery and evaluation in human Cardiomyopathies through next-generation sequencing.

Mutations in mitochondrial DNA (mtDNA) may cause maternally-inherited cardiomyopathy and heart failure. In homoplasmy all mtDNA copies contain the mutation. In heteroplasmy there is a mixture of normal and mutant copies of mtDNA. The clinical phenotype of an affected individual depends on the type of genetic defect and the ratios of mutant and normal mtDNA in affected tissues. We aimed at determining the sensitivity of next-generation sequencing compared to Sanger sequencing for mutation detection in patients with mitochondrial cardiomyopathy. We studied 18 patients with mitochondrial cardiomyopathy and two with suspected mitochondrial disease. We “shotgun” sequenced PCR-amplified mtDNA and multiplexed using a single run on Roches 454 Genome Sequencer. By mapping to the reference sequence, we obtained 1,300× average coverage per case and identified high-confidence variants. By comparing these to >400 mtDNA substitution variants detected by Sanger, we found 98% concordance in variant detection. Simulation studies showed that >95% of the homoplasmic variants were detected at a minimum sequence coverage of 20× while heteroplasmic variants required >200× coverage. Several Sanger “misses” were detected by 454 sequencing. These included the novel heteroplasmic 7501T>C in tRNA serine 1 in a patient with sudden cardiac death. These results support a potential role of next-generation sequencing in the discovery of novel mtDNA variants with heteroplasmy below the level reliably detected with Sanger sequencing. We hope that this will assist in the identification of mtDNA mutations and key genetic determinants for cardiomyopathy and mitochondrial disease.

Noncompaction of the left ventricle : primary cardiomyopathy with an elusive genetic etiology

Purpose of review Noncompaction of the left ventricle is a descriptive anatomical term and recently recognized primary cardiomyopathy. Cardiac imaging now allows for prompt detection. The specific etiology remains poorly understood, however, and the major genetic determinants are unknown. This review describes recent data showing the genetic heterogeneity and overlap with other cardiomyopathies. Understanding the genetics may depend on clarifying the distinctive diagnostic features and investigating the contribution of all known cardiomyopathy-causing genes with overlapping morphology. Recent findings Adding to the known genes (TAZ, DTNA, LDB3 and LMNA), recent work has identified SCN5A, MYH7 and MYBPC3 as associated loci. LDB3 may also be a genetic modifier. Case reports and linkage studies suggest additional loci at 1p36, 1q43 and 11p15. Aside from Barth syndrome, other genetic and metabolic syndromes with noncompaction have been described. Despite this, large studies have failed to identify the etiology in the majority of patients. Summary Despite advances in detection, comprehensive clinical, pathological, genetic, and family studies are necessary to define the phenotypic overlap with other cardiomyopathies. Without a more precise understanding of its etiology, the answers to the questions regarding the clinical relevance and management of patients with noncompaction of the left ventricle will remain elusive.

Very early complete hydatidiform mole

Recent trends toward early pregnancy ultrasound have led to evacuation of complete hydatidiform moles at a stage before the development of diffuse trophoblast hyperplasia and villous cavitation. Absence of these recognized diagnostic criteria can lead to misdiagnosis and subsequent trophoblastic neoplasia. The authors identified a case of very early complete hydatidiform mole (VECM) on review of a previous curettage specimen when the patient presented 4 weeks later with increasing human chorionic gonadotropin (HCC) titers and the typical histological features of complete mole on a subsequent curettage. DNA studies on this index case and three subsequent similar specimens confirmed the diagnosis of complete hydatidiform mole using polymerase chain reaction (PCR) amplification of eight microsatellite markers on microdissected maternal and villous tissue. VECM were compared with spontaneous abortions and elective terminations of a similar gestational age to develop diagnostic criteria. Five cardinal diagnostic features were identified: redundant bulbous terminal villi, hypercellular villous stroma, a labyrinthine network of vinous stromal canaliculi, focal cytotrophoblast and syncytiotrophoblast hyperplasia on both villi and the undersurface of the chorionic plate, and enlarged hyperchromatic implantation site trophoblast.

Severity of cardiomyopathy associated with adenine nucleotide translocator-1 deficiency correlates with mtDNA haplogroup

Mutations of both nuclear and mitochondrial DNA (mtDNA)–encoded mitochondrial proteins can cause cardiomyopathy associated with mitochondrial dysfunction. Hence, the cardiac phenotype of nuclear DNA mitochondrial mutations might be modulated by mtDNA variation. We studied a 13-generation Mennonite pedigree with autosomal recessive myopathy and cardiomyopathy due to an SLC25A4 frameshift null mutation (c.523delC, p.Q175RfsX38), which codes for the heart-muscle isoform of the adenine nucleotide translocator–1. Ten homozygous null (adenine nucleotide translocator–1−/−) patients monitored over a median of 6 years had a phenotype of progressive myocardial thickening, hyperalaninemia, lactic acidosis, exercise intolerance, and persistent adrenergic activation. Electrocardiography and echocardiography with velocity vector imaging revealed abnormal contractile mechanics, myocardial repolarization abnormalities, and impaired left ventricular relaxation. End-stage heart disease was characterized by massive, symmetric, concentric cardiac hypertrophy; widespread cardiomyocyte degeneration; overabundant and structurally abnormal mitochondria; extensive subendocardial interstitial fibrosis; and marked hypertrophy of arteriolar smooth muscle. Substantial variability in the progression and severity of heart disease segregated with maternal lineage, and sequencing of mtDNA from five maternal lineages revealed two major European haplogroups, U and H. Patients with the haplogroup U mtDNAs had more rapid and severe cardiomyopathy than those with haplogroup H.

Cytogenetic analysis of spontaneous abortions: Comparison of techniques and assessment of the incidence of confined placental mosaicism

Cytogenetic studies on spontaneous abortions traditionally have used one of two methodologies, direct preparations or long-term culture, to determine the chromosome constitution of either the cytotrophoblast or villous stroma, respectively. Few studies have utilized both techniques simultaneously to compare the relative efficiencies of each method and to assess the contribution of confined placental mosaicism (CPM). The present report summarizes cytogenetic studies on 691 consecutive spontaneous abortions using long-term culture, direct preparations, or both. All 691 cases were analyzed by long-term culture and 177 cases were analyzed using both long-term culture and direct preparations. The results indicate that the two methods have similar success rates, 82% for long-term culture and 76% for direct preparation; however, the proportion of normal females was significantly increased in the culture method, presumably attributable to maternal contamination. In 107 cases, results were obtained from both methods with 22 discrepancies identified. However, most of these involved a 46,XX result in culture, consistent with maternal contamination in the cultured preparation. Therefore, to estimate the proportion of CPM we excluded cases with a 46,XX result in culture and found four (6.1%) of the remaining 65 cases to be consistent with CPM. These cases consisted of normal or mosaic aneuploid cytotrophoblast and non-mosaic aneuploid villous stroma. These studies suggest that each method has specific advantages in the analysis of spontaneous abortions. Direct preparations are less prone to maternal contamination, but certain chromosome abnormalities are more likely to be identified using long-term culture.

Prevalence of developmental and inflammatory lesions in nonmolar first-trimester spontaneous abortions

The management of patients with first-trimester spontaneous abortions is handicapped by two problems: difficulty in recognizing conceptions that abort because of abnormal karyotypes and an incomplete understanding of what causes abortions with normal karyotypes. Our goals in this study were to define features useful in distinguishing normal from abnormal karyotype and to identify pathological processes contributing to abortions with a normal karyotype. The study population consisted of 668 well-characterized first-trimester spontaneous abortions derived from a larger study of 1,054 consecutively karyotyped spontaneous abortions. Clinical factors increased in specimens with normal karyotype were maternal age younger than 20 years (P=.0003) and autoimmune markers (P=.0474). Developmental features associated with abnormal karyotype were developmental stage less than 6 weeks (P=.0017), hydropic villi greater than 1 mm (P=.0004), and villi with two or more dysmorphic features (P=.0001). Developmental stage greater than 11.5 weeks was increased with normal karyotype (P=.0001). Histological features increased in specimens with a normal karyotype were chronic intervillositis (P=.0003), increased perivillous fibrin deposition with intermediate trophoblast (P=.0006), decidual plasma cells (P=.0040), deciduitis without plasma cells (P=.0660), and chronic villitis (P=.1581). Overall, 19% of samples with a normal karyotype versus 8% with abnormal karyotype had one or more of these findings (P < .0001). Autoimmune markers, chronic intervillositis, and increased perivillous fibrin with intermediate trophoblast all had positive predictive values greater than 85% for normal karyotype, whereas dysmorphic villi had a positive predictive value of 90% for abnormal karyotype. Patients with recurrent spontaneous abortion and normal karyotype were more likely to have one or more of the histological features listed above (31%) than patients with normal karyotype and no prior abortions (13%) and patients with recurrent abortion and abnormal karyotype (11%).

Mitochondrial cardiomyopathies: how to identify candidate pathogenic mutations by mitochondrial DNA sequencing, MITOMASTER and phylogeny.

Pathogenic mitochondrial DNA (mtDNA) mutations leading to mitochondrial dysfunction can cause cardiomyopathy and heart failure. Owing to a high mutation rate, mtDNA defects may occur at any nucleotide in its 16 569 bp sequence. Complete mtDNA sequencing may detect pathogenic mutations, which can be difficult to interpret because of normal ethnic/geographic-associated haplogroup variation. Our goal is to show how to identify candidate mtDNA mutations by sorting out polymorphisms using readily available online tools. The purpose of this approach is to help investigators in prioritizing mtDNA variants for functional analysis to establish pathogenicity. We analyzed complete mtDNA sequences from 29 Italian patients with mitochondrial cardiomyopathy or suspected disease. Using MITOMASTER and PhyloTree, we characterized 593 substitution variants by haplogroup and allele frequencies to identify all novel, non-haplogroup-associated variants. MITOMASTER permitted determination of each variants location, amino acid change and evolutionary conservation. We found that 98% of variants were common or rare, haplogroup-associated variants, and thus unlikely to be primary cause in 80% of cases. Six variants were novel, non-haplogroup variants and thus possible contributors to disease etiology. Two with the greatest pathogenic potential were heteroplasmic, nonsynonymous variants: m.15132T>C in MT-CYB for a patient with hypertrophic dilated cardiomyopathy and m.6570G>T in MT-CO1 for a patient with myopathy. In summary, we have used our automated information system, MITOMASTER, to make a preliminary distinction between normal mtDNA variation and pathogenic mutations in patient samples; this fast and easy approach allowed us to select the variants for traditional analysis to establish pathogenicity.

Studies of non-disjunction in trisomies 2, 7, 15, and 22: does the parental origin of trisomy influence placental morphology?

Recently, there have been several molecular studies of trisomic fetuses and liveborns which have examined the parent and meiotic stage of origin of nondisjunction. However, little is known about the possible phenotypic effects of the origin of trisomy. For trisomic spontaneous abortions, no distinct phenotype has been described, although some have been reported to have features, such as trophoblastic hyperplasia, similar to hydatidiform moles. In the present report, we describe molecular and histological studies of spontaneous abortions with trisomies 2, 7, 15, or 22, conditions occasionally linked to trophoblastic hyperplasia. Our results provide strong evidence for chromosome specific mechanisms of nondisjunction, with trisomy 2 having a high frequency of paternally derived cases and trisomy 7 typically originating postzygotically. In studies correlating parental origin of trisomy with phenotype, we found no difference in the proportion of cases with trophoblastic hyperplasia, fetal tissue, nucleated red blood cells, or hydropic villi among paternally or maternally derived trisomies 2, 7, 15, or 22. However, paternally derived trisomies tended to abort earlier than maternally derived trisomies. This suggests that parental origin might affect the developmental stage at which abortion occurs but not other features of placental phenotype.