Roger V. Lebo

ACMG position statement on prenatal/preconception expanded carrier screening.

For years, clinicians have offered gene-by-gene carrier screening to patients and couples considering future pregnancy or those with an ongoing pregnancy early in gestation. Examples include ethnic-specific screening offered to Ashkenazi Jewish patients and panethnic screening for cystic fibrosis and spinal muscular atrophy. Next-generation sequencing methods now available permit screening for many more disorders with high fidelity, quick turnaround time, and lower costs. However, instituting these technologies carries with it perils that must be addressed. The basis for the selection of disorders on expanded carrier screening panels should be disclosed. The information provided about disorders with mild phenotypes, variable expression, low penetrance, and/or characterized by an adult onset should be complete and transparent, allowing patients to opt out of receiving these test results. Patients also must be made aware of the concept of residual risk following negative test results. Laboratories have a duty to participate in and facilitate this information transfer.Genet Med 2013:15(6):482–483

Developing a sustainable process to provide quality control materials for genetic testing

Purpose: To provide a summary of the outcomes of two working conferences organized by the Centers for Disease Control and Prevention (CDC), to develop recommendations for practical, sustainable mechanisms to make quality control (QC) materials available to the genetic testing community.Methods: Participants were selected to include experts in genetic testing and molecular diagnostics from professional organizations, government agencies, industry, laboratories, academic institutions, cell repositories, and proficiency testing (PT)/external Quality Assessment (EQA) programs. Current efforts to develop QC materials for genetic tests were reviewed; key issues and areas of need were identified; and workgroups were formed to address each area of need and to formulate recommendations and next steps.Results: Recommendations were developed toward establishing a sustainable process to improve the availability of appropriate QC materials for genetic testing, with an emphasis on molecular genetic testing as an initial step.Conclusions: Improving the availability of appropriate QC materials is of critical importance for assuring the quality of genetic testing, enhancing performance evaluation and PT/EQA programs, and facilitating new test development. To meet the needs of the rapidly expanding capacity of genetic testing in clinical and public health settings, a comprehensive, coordinated program should be developed. A Genetic Testing Quality Control Materials Program has therefore been established by CDC in March 2005 to serve these needs.

Objective aneuploidy detection for fetal and neonatal screening using comparative genomic hybridization (CGH)

Comparative genomic hybridization (CGH) allows entire genomes to be scanned for whole and segmental aneuploidy and thus may be an appropriate tool for the detection of clinically important abnormalities during fetal and neonatal screening. Criteria to distinguish between significant aberrations and experimental artifacts are essential for these applications. This report describes the use of a t-statistic to detect changes in CGH profiles that differ significantly from variations that occur in CGH profiles of normal samples. Eleven cell lines derived from fetal or neonatal patients were analyzed in this study. Aneuploidies in these lines included trisomies for chromosomes 13, 16, 18, and 21 and monosomy for distal 5p and tetrasomy 18p. Aneuploidy was detected in all samples by using the t-statistic, although the extent of the aneuploid region was not correctly estimated in some cases. A detailed description of the t-statistic fused for making these CGH comparisons is described in a companion paper (Moore et al., Cytometry 28:183-190, 1997.

True trisomy 2 mosaicism in amniocytes and newborn liver associated with multiple system abnormalities

Among 58,000 amniocenteses completed, our laboratories found one case of true cytogenetic trisomy 2 mosaicism in a fetus with multiple abnormalities. In contrast, 11 fetuses phenotypically normal at birth were found to have true trisomy 2 mosaicism in their chorionic villus cells among the 10,500 fetuses tested by chorionic villus sampling (CVS). In our single abnormal case, amniocentesis performed at 19 weeks after finding an elevated maternal serum AFP found two independent cultures with trisomy 2 karyotypes in 8 of 25 and 7 of 31 amniocytes, respectively. Although oligohydramnios was noted by ultrasound, the mother elected to continue the pregnancy. At 26 weeks the fetus had intrauterine growth retardation (IUGR), hydronephrosis, and cardiac abnormalities. When delivered by Cesarean section at 30 weeks, the infant had multiple anomalies and developed necrotizing enterocolitis and severe cholestasis. At 5 months coronal magnetic resonance imaging (MRI) displayed delayed myelination and abnormal brain morphology. The patient also exhibited significant growth failure and developmental delay. Although chromosomes were normal in blood, skin fibroblasts, and ascites fluid cells, 4 of 100 hepatic biopsy fibroblasts were 47,XY,+2. Molecular analysis excluded uniparental disomy (UPD) of chromosome 2 in the 46,XY cell line. This and other reports of rare phenotypically abnormal trisomy 2 mosaic fetuses identified by karyotyping amniocytes emphasizes the substantially higher fetal risk of abnormal development than when trisomy 2 is found only in chorionic villus cells.

The shorter zinc finger protein ZNF230 gene message is transcribed in fertile male testes and may be related to human spermatogenesis

The zinc finger gene family represents one of the largest in the mammalian genome, with several of these genes reported to be involved in spermatogenesis. A newly discovered gene has been identified that is expressed abundantly in the testicular tissue of fertile men as determined by mRNA differential display. The gene encodes a C(3)HC(4)-type zinc finger protein motif (ring finger motif) consistent with a role in pre-meiotic or post-meiotic sperm development. The gene was named ZNF230 and mapped to the short arm of chromosome 11 (11p15). ZNF230 has two transcripts, of 1 kb and 4.4 kb in length. The shorter 1 kb transcript was only detected in testicular tissue whereas the longer 4.4 kb transcript was not detected in testis but was found in several other tissues. The lack of detectable ZNF230 expression in azoospermic patients by reverse transcriptase-mediated PCR analysis is interpreted to mean that this gene is involved in maintaining normal human male fertility.

Mutation Analysis in Rett Syndrome

Rett syndrome is an X-linked dominant neurodevelopmental disorder caused by mutations in the MECP2 gene. Mutations have been demonstrated in more than 80% of females with typical features of Rett syndrome. We identified mutations in the MECP2 gene and documented the clinical manifestations in 65 Rett syndrome patients to characterize the genotype-phenotype spectrum. Bidirectional sequencing of the entire MECP2 coding region was performed. We diagnosed 65 patients with MECP2 mutations. Of these, 15 mutations had been reported previously and 13 are novel. Two patients have multiple deletions within the MECP2 gene. Eight common mutations were found in 43 of 65 patients (66.15%). The majority of patients with identified mutations have the classic Rett phenotype, and several had atypical phenotypes. MECP2 analysis identified mutations in almost all cases of typical Rett syndrome, as well as in some with atypical phenotypes. Eleven (20.4%) of the 54 patients with defined mutations and in whom phenotypic data were obtained did not develop acquired microcephaly. Hence, microcephaly at birth or absence of acquired microcephaly does not obviate the need for MECP2 analysis. We have initiated cascade testing starting with PCR analysis for common mutations followed by sequencing, when necessary. Analysis of common mutations before sequencing the entire gene is anticipated to be the most efficacious strategy to identify Rett syndrome gene mutations.

Rett syndrome from quintuple and triple deletions within the MECP2 deletion hotspot region.

Rett syndrome results from mutations in the X‐linked methyl‐CpG‐binding protein 2 (MECP2) gene, which are nearly always lethal in males and lead to regression and reduced life expectancy in females. Herein we report one propositus with five tandem deletions and a second propositus with three tandem deletions within MECP2 exon 4 that encode truncated protein products resulting in classic Rett syndrome. These deletion breakpoints and single deletions in 3 other patients were all found within a 185‐bp region along with 64 of 69 other reported deletion breakpoints in the MECP2 gene. Illegitimate recombination resulting in deletion at a substantial proportion of the shared MECP2 sites is enhanced by repeated guanosine (G) DNA sequences in the antisense direction, consistent with reports at other gene loci that polypurine (multiple guanosine or adenosine (A)) basepairs enhance sequence deletion. Multiple deletions at the same poly G recombination sites confirm the existence of deletion hotspots in this gene region with numerous repeated antisense sites that are enriched 26‐ to 161‐fold. Deletion by illegitimate recombination within a single allele can occur during mitotic or meiotic cell cycles. Although prone to disease‐causing deletion, this region is unique in humans and highly conserved among mammals for the last 75 000 000 years to maintain the MECP2 genes critical function.

Tandem duplication/deletion in a maternally derived chromosome 9 supernumerary derivative resulting in 9p trisomy and partial 9q tetrasomy

A 19-week stillborn female fetus with bilateral cleft palate, horseshoe kidney, bicornuate uterus, low-set ears, and intrauterine growth retardation (IUGR) was found to have a supernumerary derivative chromosome 9 (der(9)) with an apparent tandem duplication in the long arm. PCR analysis at five polymorphic loci confirmed the duplication and showed an adjacent deletion, while whole chromosome FISH demonstrated only chromosome 9 to be involved. Further FISH studies of der(9) found the 9qh region to be duplicated, telomeric sequences to be intact, and subtelomeric sequences to be absent. Thus, the fetus was determined to be trisomic for 9pter-->9q12 and 9q34.3-->9qter, tetrasomic for 9q12--> 9q33, and disomic for 9q33-->9q34.3. These results are consistent with a tandem duplication of 9q12-->9q33 and adjacent distal deletion as designated by the karyotype, 47,XX,+der(9)dup(9) (q12q33)del(9) (q33q34.3).ish der(9)(WCP9+,D9Z1x2,STP9q-, AHT+) de novo. In addition to characterizing der(9), the combined PCR and cytogenetic studies refined the Genome Database Map of three loci (D9S907, D9S155, and D9S302) approximately to the distal 9q33 region. Without the attempt to refine breakpoints by PCR analysis, the deletion in distal 9q would not have been detected. Tandem direct duplication/deletion chromosomes have been reported in fewer cases than inverted duplication/deletions. We propose mechanisms of origin, consistent with those for recurrent inter stitial microdeletion and microduplication syndromes, shown to arise by recombination at homologous, flanking DNA sequences.

Rare Etiology of Autosomal Recessive Disease in a Child with Noncarrier Parents

A child with maple syrup urine disease type 2 (MSUD2) was found to be homozygous for a 10-bp MSUD2-gene deletion on chromosome 1. Both purported parents were tested, and neither carries the gene deletion. Polymorphic simple-sequence repeat analyses at 15 loci on chromosome 1 and at 16 loci on other chromosomes confirmed parentage and revealed that a de novo mutation prior to maternal meiosis I, followed by nondisjunction in maternal meiosis II, resulted in an oocyte with two copies of the de novo mutant allele. Fertilization by a sperm that did not carry a paternal chromosome 1 or subsequent mitotic loss of the paternal chromosome 1 resulted in the propositus inheriting two mutant MSUD2 alleles on two maternal number 1 chromosomes.

Symmetric replication of an unstable isodicentric Xq chromosome derived from isolocal maternal sister chromatid recombination

An amniocyte culture was found to be mosaic for 45,X/46,X, idic(X)(p11.2)/ 47,X, idic(X)(p11.2),idic(X)(p11.2) cell lines, reflecting mitotic nondisjunction of the idic(X)(p11.2) chromosome. Upon learning of abnormal karyotype and ultrasound findings, the parents decided to discontinue the pregnancy. Subsequent cultures of fetal skin, kidney, and lung were mosaic 45,X/46,X,idic(X)(p11.2) reflecting mitotic loss of the unstable idic(X)(p11.2) chromosome. C-banding and in situ hybridization of X chromosome-specific alpha-satellite probe to metaphase fetal cells confirmed two centromeres on the idic(X)(p11.2) chromosome with both centromeres appearing to be active in two-thirds of cells. This result was confirmed by centromere protein-E (CENP-E) antibody staining which delineated 80% of scored cells with two active centromeres and 20% with 1 active centromere. Bromodeoxyuridine (BrdU) incorporation and acridine orange staining characterized the DNA replication pattern of the idic(X)(p11.2) chromosome as late and symmetrically replicating. Polymerase chain reaction analysis of highly polymorphic loci determined that the normal X chromosome carried paternal alleles and the idic(X)(p11.2) chromosome carried maternal alleles from only one grandparental chromosome. Overall, the results suggest that recombination occurred between two maternal sister chromatids both in the same chromosome band Xp11.2 (isolocal) prior to maternal meiosis II anaphase to generate an unstable maternal idic(X)(p11.2) chromosome. Additional factors that could contribute to i(Xq) and idic(X) formation and instability are discussed along with a mechanism to explain the high frequency of intrauterine loss in 45,X pregnancies.