Carrier screening in the Mexican Jewish community using a pan-ethnic expanded carrier screening NGS panel.

PURPOSE: The Mexican Jewish community (MJC) is a previously uncharacterized, genetically isolated group composed of Ashkenazi and Sephardi-Mizrahi Jews who migrated in the early 1900s. We aimed to determine the heterozygote frequency of disease-causing variants in 302 genes in this population. METHODS: We conducted a cross-sectional study of the MJC involving individuals representing Ashkenazi Jews, Sephardi-Mizrahi Jews, or mixed-ancestry Jews. We offered saliva-based preconception pan-ethnic expanded carrier screening, which examined 302 genes. We analyzed heterozygote frequencies of pathogenic/likely pathogenic variants and compared them with those in the Genome Aggregation Database (gnomAD). RESULTS: We recruited 208 participants. The carrier screening results showed that 72.1% were heterozygous for at least 1 severe disease-causing variant in 1 of the genes analyzed. The most common genes with severe disease-causing variants were CFTR (16.8% of participants), MEFV (11.5%), WNT10A (6.7%), and GBA (6.7%). The allele frequencies were compared with those in the gnomAD; 85% of variant frequencies were statistically different from those found in gnomAD (P <.05). Finally, 6% of couples were at risk of having a child with a severe disorder. CONCLUSION: The heterozygote frequency of at least 1 severe disease-causing variant in the MJC was 72.1%. The use of carrier screening in the MJC and other understudied populations could help parents make more informed decisions.

Specifications of the ACMG/AMP standards and guidelines for mitochondrial DNA variant interpretation.

Mitochondrial DNA (mtDNA) variant pathogenicity interpretation has special considerations given unique features of the mtDNA genome, including maternal inheritance, variant heteroplasmy, threshold effect, absence of splicing, and contextual effects of haplogroups. Currently, there are insufficient standardized criteria for mtDNA variant assessment, which leads to inconsistencies in clinical variant pathogenicity reporting. An international working group of mtDNA experts was assembled within the Mitochondrial Disease Sequence Data Resource Consortium and obtained Expert Panel status from ClinGen. This group reviewed the 2015 American College of Medical Genetics and Association of Molecular Pathology standards and guidelines that are widely used for clinical interpretation of DNA sequence variants and provided further specifications for additional and specific guidance related to mtDNA variant classification. These Expert Panel consensus specifications allow for consistent consideration of the unique aspects of the mtDNA genome that directly influence variant assessment, including addressing mtDNA genome composition and structure, haplogroups and phylogeny, maternal inheritance, heteroplasmy, and functional analyses unique to mtDNA, as well as specifications for utilization of mtDNA genomic databases and computational algorithms.

How I curate: applying American Society of Hematology-Clinical Genome Resource Myeloid Malignancy Variant Curation Expert Panel rules for RUNX1 variant curation for germline predisposition to myeloid malignancies.

The broad use of next-generation sequencing and microarray platforms in research and clinical laboratories has led to an increasing appreciation of the role of germline mutations in genes involved in hematopoiesis and lineage differentiation that contribute to myeloid neoplasms. Despite implementation of the American College of Medical Genetics and Genomics and Association for Molecular Pathology 2015 guidelines for sequence variant interpretation, the number of variants deposited in ClinVar, a genomic repository of genotype and phenotype data, and classified as having uncertain significance or being discordantly classified among clinical laboratories remains elevated and contributes to indeterminate or inconsistent patient care. In 2018, the American Society of Hematology and the Clinical Genome Resource co-sponsored the Myeloid Malignancy Variant Curation Expert Panel to develop rules for classifying gene variants associated with germline predisposition to myeloid neoplasia. Herein, we demonstrate application of our rules developed for the RUNX1 gene to variants in six examples to show how we would classify them within the proposed framework.

Variable cardiovascular phenotypes associated with SMAD2 pathogenic variants.

SMAD2 is a downstream effector in the TGF-ß signaling pathway, which is important for pattern formation and tissue differentiation. Pathogenic variants in SMAD2 have been reported in association with arterial aneurysms and dissections and in large cohorts of subjects with complex congenital heart disease (CHD). We used whole exome sequencing (WES) to investigate the molecular cause of CHD and other congenital anomalies in three probands and of an arterial aneurysm in an additional patient. Patients 1 and 2 presented with complex CHD, developmental delay, seizures, dysmorphic features, short stature, and poor weight gain. Patient 3 was a fetus with complex CHD and heterotaxy. The fourth patient is an adult female with aortic root aneurysm and physical features suggestive of a connective tissue disorder. WES identified pathogenic truncating variants, a splice variant, and a predicted deleterious missense variant in SMAD2. We compare the phenotypes and genotypes in our patients with previously reported cases. Our data suggest two distinct phenotypes associated with pathogenic variants in SMAD2: complex CHD with or without laterality defects and other congenital anomalies, and a late-onset vascular phenotype characterized by arterial aneurysms with connective tissue abnormalities.

Cytogenetics and holoprosencephaly: A chromosomal microarray study of 222 individuals with holoprosencephaly.

Holoprosencephaly (HPE), a common developmental forebrain malformation, is characterized by failure of the cerebrum to completely divide into left and right hemispheres. The etiology of HPE is heterogeneous and a number of environmental and genetic factors have been identified. Cytogenetically visible alterations occur in 25% to 45% of HPE patients and cytogenetic techniques have long been used to study copy number variants (CNVs) in this disorder. The karyotype approach initially demonstrated several recurrent chromosomal anomalies, which led to the identification of HPE-specific loci and, eventually, several major HPE genes. More recently, higher-resolution cytogenetic techniques such as subtelomeric multiplex ligation-dependent probe amplification and chromosomal microarray have been used to analyze chromosomal anomalies. By using chromosomal microarray, we sought to identify submicroscopic chromosomal deletions and duplications in patients with HPE. In an analysis of 222 individuals with HPE, a deletion or duplication was detected in 107 individuals. Of these 107 individuals, 23 (21%) had variants that were classified as pathogenic or likely pathogenic by board-certified medical geneticists. We identified multiple patients with deletions in established HPE loci as well as three patients with deletions encompassed by 6q12-q14.3, a CNV previously reported by Bendavid et al. In addition, we identified a new locus, 16p13.2 that warrants further investigation for HPE association. Incidentally, we also found a case of Potocki-Lupski syndrome, a case of Phelan-McDermid syndrome, and multiple cases of 22q11.2 deletion syndrome within our cohort. These data confirm the genetically heterogeneous nature of HPE, and also demonstrate clinical utility of chromosomal microarray in diagnosing patients affected by HPE.

Pathogenic and likely pathogenic variant prevalence among the first 10,000 patients referred for next-generation cancer panel testing.

PURPOSE: Germ-line testing for panels of cancer genes using next-generation sequencing is becoming more common in clinical care. We report our experience as a clinical laboratory testing both well-established, high-risk cancer genes (e.g., BRCA1/2, MLH1, MSH2) as well as more recently identified cancer genes (e.g., PALB2, BRIP1), many of which have increased but less well-defined penetrance. METHODS: Clinical genetic testing was performed on over 10,000 consecutive cases referred for evaluation of germ-line cancer genes, and results were analyzed for frequency of pathogenic or likely pathogenic variants, and were stratified by testing panel, gene, and clinical history. RESULTS: Overall, a molecular diagnosis was made in 9.0% of patients tested, with the highest yield in the Lynch syndrome/colorectal cancer panel. In patients with breast, ovarian, or colon/stomach cancer, positive yields were 9.7, 13.4, and 14.8%, respectively. Approximately half of the pathogenic variants identified in patients with breast or ovarian cancer were in genes other than BRCA1/2. CONCLUSION: The high frequency of positive results in a wide range of cancer genes, including those of high penetrance and with clinical care guidelines, underscores both the genetic heterogeneity of hereditary cancer and the usefulness of multigene panels over genetic tests of one or two genes.Genet Med 18 8, 823-832.

Cascade testing for hereditary cancer: comprehensive multigene panels identify unexpected actionable findings in relatives.

Current guidelines recommend single variant testing in relatives of patients with known pathogenic or likely pathogenic germline variants in cancer predisposition genes. This approach may preclude the use of risk-reducing strategies in family members who have pathogenic or likely pathogenic germline variants in other cancer predisposition genes. Cascade testing using multigene panels was performed in 3696 relatives of 7433 probands. Unexpected pathogenic or likely pathogenic germline variants were identified in 230 (6.2%) relatives, including 144 who were negative for the familial pathogenic or likely pathogenic variant but positive for a pathogenic or likely pathogenic variant in a different gene than the proband and 74 who tested positive for the familial pathogenic or likely pathogenic variant and had an additional pathogenic or likely pathogenic variant in a different gene than the proband. Of the relatives with unexpected pathogenic or likely pathogenic germline variants, 36.3% would have qualified for different or additional cancer screening recommendations. Limiting cascade testing to only the familial pathogenic or likely pathogenic variant would have resulted in missed, actionable findings for a subset of relatives.

Genotypic and phenotypic analysis of 396 individuals with mutations in Sonic Hedgehog.

BACKGROUND: Holoprosencephaly (HPE), the most common malformation of the human forebrain, may result from mutations in over 12 genes. Sonic Hedgehog (SHH) was the first such gene discovered; mutations in SHH remain the most common cause of non-chromosomal HPE. The severity spectrum is wide, ranging from incompatibility with extrauterine life to isolated midline facial differences. OBJECTIVE: To characterise genetic and clinical findings in individuals with SHH mutations. METHODS: Through the National Institutes of Health and collaborating centres, DNA from approximately 2000 individuals with HPE spectrum disorders were analysed for SHH variations. Clinical details were examined and combined with published cases. RESULTS: This study describes 396 individuals, representing 157 unrelated kindreds, with SHH mutations; 141 (36%) have not been previously reported. SHH mutations more commonly resulted in non-HPE (64%) than frank HPE (36%), and non-HPE was significantly more common in patients with SHH than in those with mutations in the other common HPE related genes (p<0.0001 compared to ZIC2 or SIX3). Individuals with truncating mutations were significantly more likely to have frank HPE than those with non-truncating mutations (49% vs 35%, respectively; p=0.012). While mutations were significantly more common in the N-terminus than in the C-terminus (including accounting for the relative size of the coding regions, p=0.00010), no specific genotype-phenotype correlations could be established regarding mutation location. CONCLUSIONS: SHH mutations overall result in milder disease than mutations in other common HPE related genes. HPE is more frequent in individuals with truncating mutations, but clinical predictions at the individual level remain elusive.

Missense substitutions in the GAS1 protein present in holoprosencephaly patients reduce the affinity for its ligand, SHH.

Holprosencephaly (HPE) is the most common disorder of the developing forebrain in humans, and is characterized by varying degrees of abnormal union of the cerebral hemispheres. These defects are typically co-associated with midline craniofacial anomalies. The combination of forebrain and craniofacial defects that comprise HPE can present along a broad and variable phenotypic spectrum. Both the SHH and NODAL signaling pathways play important roles in the pathogenesis of this disorder. Disruption of these pathways by chromosomal rearrangements, mutations in pathway-related genes and/or biochemical alterations are proposed to contribute to HPE in a large number of patients. Additional factors that are not yet fully delineated are also very likely to be involved in the pathogenesis and phenotypic heterogeneity of the disorder. Genetic loss of GAS1, a cell membrane receptor and positive regulator of SHH, has been demonstrated to contribute to the HPE phenotypic spectrum in animal models. We have evaluated the coding and flanking sequence of GAS1 in 394 patients who have clinical findings within the HPE phenotypic spectrum, and now report five novel missense sequence variants among five unrelated HPE probands. Finally, we tested the effect of these variants (as well as previously reported GAS1 variants) on the ability of GAS1 to bind to SHH. Here, we demonstrate that sequence variants in GAS1 can impair its physical interaction with SHH, suggesting a decrease in the SHH downstream signaling cascade as a pathogenic mechanism of disease.

Comparison of mutation findings in ZIC2 between microform and classical holoprosencephaly in a Brazilian cohort.

BACKGROUND: Holoprosencephaly is the most frequent congenital malformation of the forebrain in humans. It is anatomically classified by the relative degree of abnormal formation and separation of the developing central nervous system. Mutations of ZIC2 are the second most common heterozygous variations detected in holoprosencephaly (HPE) patients. Mutations in most known HPE genes typically result in variable phenotypes that rage from classic alobar HPE to microforms represented by hypotelorism, solitary central maxillary incisor (SCMI), and cleft lip/palate, among others. Patients with HPE owing to ZIC2 mutations have recently been described by a distinct phenotype compared with mutations in other HPE causative genes. METHODS: We report the comparison of ZIC2 molecular findings by Sanger bidirectional DNA sequencing and ad hoc genotyping in a cohort of 105 Brazilian patients within the clinical spectrum of HPE, including classic and microform groups. RESULTS: We detected a total of five variants in the ZIC2 gene: a common histidine tract expansion c.716_718dup (p.His239dup), a rare c.1377_1391del_homozygous (p.Ala466_470del, or Ala 15 to 10 contraction), a novel intronic c.1239+18G>A variant, a novel frameshift c.1215dupC (p.Ser406Glnfs*11), and a c.1401_1406dup (p.Ala469_470dup, or alanine tract expansion to 17 residues). CONCLUSIONS: From these patients, only the latter two mutations found in classic HPE are likely to be medically significant. In contrast, variants detected in the microform group are not likely to be pathogenic. We show conclusively that the histidine tract expansion is a polymorphic alteration that demonstrates considerable differences in allele frequencies across different ethnic groups. Therefore, careful population studies of rare variants can improve genotype-phenotype correlations. Birth Defects Research (Part A) 2012.

Germline Pathogenic Variant Prevalence Among Latin American and US Hispanic Individuals Undergoing Testing for Hereditary Breast and Ovarian Cancer: A Cross-Sectional Study.

PURPOSE: To report on pathogenic germline variants detected among individuals undergoing genetic testing for hereditary breast and/or ovarian cancer (HBOC) from Latin America and compare them with self-reported Hispanic individuals from the United States. METHODS: In this cross-sectional study, unrelated individuals with a personal/family history suggestive of HBOC who received clinician-ordered germline multigene sequencing were grouped according to the location of the ordering physician: group A, Mexico, Central America, and the Caribbean; group B, South America; and group C, United States with individuals who self-reported Hispanic ethnicity. Relatives who underwent cascade testing were analyzed separately. RESULTS: Among 24,075 unrelated probands across all regions, most were female (94.9%) and reported a personal history suggestive of HBOC (range, 65.0%-80.6%); the mean age at testing was 49.1 ± 13.1 years. The average number of genes analyzed per patient was highest in group A (A 63 ± 28, B 56 ± 29, and C 40 ± 28). Between 9.1% and 18.7% of patients had pathogenic germline variants in HBOC genes (highest yield in group A), with the majority associated with high HBOC risk. Compared with US Hispanics individuals the overall yield was significantly higher in both Latin American regions (A v C P = 1.64×10-9, B v C P < 2.2×10-16). Rates of variants of uncertain significance were similar across all three regions (33.7%-42.6%). Cascade testing uptake was low in all regions (A 6.6%, B 4.5%, and C 1.9%). CONCLUSION: This study highlights the importance of multigene panel testing in Latin American individuals with newly diagnosed or history of HBOC, who can benefit from medical management changes including targeted therapies, eligibility to clinical trials, risk-reducing surgeries, surveillance and prevention of secondary malignancy, and genetic counseling and subsequent cascade testing of at-risk relatives.

Personalized genomic medicine: lessons from the exome.

While genomic sequencing methods are powerful tools in the discovery of the genetic underpinnings of human disease, incidentally-revealed novel genomic risk factors may be equally important, both scientifically, and as relates to direct patient care. We performed whole-exome sequencing on a child with VACTERL association who suffered severe post-surgical neonatal pulmonary hypertension, and identified a potential novel genetic risk factor for this complication: a heterozygous mutation in CPSI. Newborn screening results from this patient's monozygotic twin provided evidence that this mutation, in combination with an environmental trigger (in this case, surgery), may have resulted in pulmonary artery hypertension due to inadequate nitric oxide production. Identification of this genetic risk factor allows for targeted medical preventative measures in this patient as well as relatives with the same mutation, and illustrates the power of incidental medical information unearthed by whole-exome sequencing.

Minimal evidence for a direct involvement of twisted gastrulation homolog 1 (TWSG1) gene in human holoprosencephaly.

Holoprosencephaly (HPE) is the most common disorder of human forebrain and facial development. Presently understood etiologies include both genetic and environmental factors, acting either alone, or more likely, in combination. The majority of patients without overt chromosomal abnormalities or recognizable associated syndromes have unidentified etiologies. A potential candidate gene, Twisted Gastrulation Homolog 1 (TWSG1), was previously suggested as a contributor to the complex genetics of human HPE based on (1) cytogenetic studies of patients with 18p deletions, (2) animal studies of TWSG1 deficient mice, and (3) the relationship of TWSG1 to bone morphogenetic protein (BMP) signaling, which modulates the primary pathway implicated in HPE, Sonic Hedgehog (SHH) signaling. Here we present the first analysis of a large cohort of patients with HPE for coding sequence variations in TWSG1. We also performed fine mapping of 18p for a subset of patients with partial 18p deletions. Surprisingly, minimal evidence for alterations of TWSG1 was found, suggesting that sequence alterations of TWSG1 are neither a common direct cause nor a frequent modifying factor for human HPE pathologies.

A broad range of ophthalmologic anomalies is part of the holoprosencephaly spectrum.

Holoprosencephaly (HPE) is the most common disorder of the developing forebrain in humans, and is characterized by failed or incomplete cleavage of the cerebral hemispheres and deep brain structures. HPE includes wide phenotypic variability, with a continuum of both brain and craniofacial anomalies. While "classic" eye findings, including the spectrum of midline anomalies ranging from cyclopia to hypotelorism, as well as chorioretinal coloboma and microphthalmia, have been frequently described in patients with HPE, other subtle eye anomalies may also occur. In our study we prospectively analyzed a small cohort of 10 patients in whom we identified mutations in SHH, SIX3, ZIC2, or FGF8, the latter of which is a very recently described HPE-associated gene. We found that 9 of 10 patients had at least two ophthalmologic anomalies, including refractive errors, microcornea, microphthalmia, blepharoptosis, exotropia, and uveal coloboma. These findings contribute to the understanding of the phenotypic variability of the HPE spectrum, and highlight findings in one medically important but often incompletely investigated system.

VACTERL association and mitochondrial dysfunction.

BACKGROUND: VACTERL association includes the presence of malformations affecting the vertebrae, anus, heart, trachea and esophagus, kidneys, and limbs. The causes remain largely unknown, but rare patients with mitochondrial dysfunction have been reported. Although clinical signs and symptoms consistent with possible mitochondrial disease are not uncommon in patients with VACTERL association, the necessary testing to confirm mitochondrial dysfunction is infrequently performed. METHODS: We describe a patient with relatively classic signs of VACTERL association who had an onset of clinical signs of mitochondrial dysfunction at 13 months of age. These signs included progressive muscle weakness, autonomic dysregulation, episodic hypoglycemia, and exocrine pancreatic dysfunction. The patient was later shown to have evidence of mitochondrial dysfunction (cytochrome c oxidase deficiency). CONCLUSIONS: Abnormal mitochondrial function may be associated with VACTERL association, and clinicians who encounter patients with VACTERL association should have a low threshold for considering mitochondrial dysfunction.

De novo deletion of chromosome 20q13.33 in a patient with tracheo-esophageal fistula, cardiac defects and genitourinary anomalies implicates GTPBP5 as a candidate gene.

BACKGROUND: Tracheo-esophageal fistula (TEF) with/or without esophageal atresia (EA) is a common congenital malformation that is often accompanied by other anomalies. The causes of this condition are thought to be heterogeneous but are overall not well understood. CASE REPORT: We identified a patient with a TEF/EA, as well as cardiac and genitourinary anomalies, who was found to have a 0.7 Mb de novo deletion of chromosome 20q13.33. One gene within the deleted interval, GTPBP5, is of particular interest as a candidate gene. CONCLUSIONS: GTPBP5 bears further study as a cause of TEF/EA accompanied by other malformations.

Mutations in ZIC2 in human holoprosencephaly: description of a novel ZIC2 specific phenotype and comprehensive analysis of 157 individuals.

BACKGROUND: Holoprosencephaly (HPE), the most common malformation of the human forebrain, may be due to mutations in genes associated with non-syndromic HPE. Mutations in ZIC2, located on chromosome 13q32, are a common cause of non-syndromic, non-chromosomal HPE. OBJECTIVE: To characterise genetic and clinical findings in patients with ZIC2 mutations. METHODS: Through the National Institutes of Health and collaborating centres, DNA from approximately 1200 individuals with HPE spectrum disorders was analysed for sequence variations in ZIC2. Clinical details were examined and all other known cases of mutations in ZIC2 were included through a literature search. RESULTS: By direct sequencing of DNA samples of an unselected group of unrelated patients with HPE in our NIH laboratory, ZIC2 mutations were found in 8.4% (49/582) of probands. A total of 157 individuals from 119 unrelated kindreds are described, including 141 patients with intragenic sequence determined mutations in ZIC2. Only 39/157 patients have previously been clinically described. Unlike HPE due to mutations in other genes, most mutations occur de novo and the distribution of HPE types differs significantly from that of non-ZIC2 related HPE. Evidence is presented for the presence of a novel facial phenotype which includes bitemporal narrowing, upslanting palpebral fissures, a short nose with anteverted nares, a broad and well demarcated philtrum, and large ears. CONCLUSIONS: HPE due to ZIC2 mutations is distinct from that due to mutations in other genes. This may shed light on the mechanisms involved in formation of the forebrain and face and will help direct genetic counselling and diagnostic strategies.

Current recommendations for the molecular evaluation of newly diagnosed holoprosencephaly patients.

Holoprosencephaly (HPE) is the most common structural malformation of the developing forebrain in humans and is typically characterized by different degrees of hemispheric separation that are often accompanied by similarly variable degrees of craniofacial and midline anomalies. HPE is a classic example of a complex genetic trait with "pseudo"-autosomal dominant transmission showing incomplete penetrance and variable expressivity. Clinical suspicion of HPE is typically based upon compatible craniofacial findings, the presence of developmental delay or seizures, or specific endocrinological abnormalities, and is then followed up by confirmation with brain imaging. Once a clinical diagnosis is made, a thorough genetic evaluation is necessary. This usually includes analysis of chromosomes by high-resolution karyotyping, clinical assessment to rule-out well recognized syndromes that are associated with HPE (e.g., Pallister-Hall syndrome, Smith-Lemli-Opitz syndrome and others), and molecular studies of the most common HPE associated genes (e.g., SHH, ZIC2 and SIX3). In this review, we provide current step-by-step recommendations that are medically indicated for the genetic evaluation of patients with newly diagnosed HPE. Moreover, we provide a brief review of several available methods used in molecular diagnostics of HPE and describe the advantages and limitations of both currently available and future tests as they relate to high throughput screening, cost, and the results that they may provide.

Holoprosencephaly flashcards: A summary for the clinician.

This material contains general information regarding the approach to patients with holoprosencephaly. For more detailed discussion, please refer to specific articles in this issue.

Analysis of genotype-phenotype correlations in human holoprosencephaly.

Since the discovery of the first gene causing holoprosencephaly (HPE), over 500 patients with mutations in genes associated with non-chromosomal, non-syndromic HPE have been described, with detailed descriptions available in over 300. Comprehensive clinical analysis of these individuals allows examination for the presence of genotype-phenotype correlations. These correlations allow a degree of differentiation between patients with mutations in different HPE-associated genes and for the application of functional studies to determine intragenic correlations. These early correlations are an important advance in the understanding of the clinical aspects of this disease, and in general argue for continued analysis of the genetic and clinical findings of large cohorts of patients with rare diseases in order to better inform both basic biological insight and care and counseling for affected patients and families.