PANGEN: an online platform for the comparison and creation of diagnostic gene panels.

Targeted gene panel sequencing is used to limit the search for causative genetic variants solely to genes with an established association with the phenotype. The design of gene panels is challenging due to the lack of consensus regarding phenotypic associations for some genes, which results in high variation in gene composition for the same panel offered by different laboratories. We developed PANGEN, a platform that provides a centralized resource for gene panel information, with the ability to compare and generate new intelligent diagnostic panels. Gene-phenotype associations were collected from 12 public and commercial sources (Blueprint, Cegat, Centogene, ClinGen, Fulgent, GeneDx, Health in Code, Human Phenotype Ontology, Invitae, PanelApp, Prevention genetics, and Pronto diagnostics). Gene-phenotype associations are categorized into tiers according to categories derived from the original source panel. Pairwise panel similarity was calculated by dividing the number of common genes by the total number of genes in both panels. Regions with extreme guanine-cytosine (GC) content were collected from the Genome in a Bottle stratifications dataset, and putative genomic duplications were retrieved from the University of Santa Cruz database. Overall, 1533 panels, 9759 phenotypes, and 6979 genes were collected. The platform provides an interface to (i) explore and compare collected panels, (ii) find similar panels, (iii) identify genes with high GC content or duplication levels, (iv) generate gene panels by combining panels from various sources, and (v) stratify a generated panel into genes with a strong phenotype association ('core') and those with a weaker association ('extended'). The presented platform represents a unique resource for gene panel exploration and comparison that facilitates the generation of tailored diagnostic panels through a public online web server. Database URL: https://c-gc.shinyapps.io/PANGEN/.

Clinical Updates and Surveillance Recommendations for DNA Replication Repair Deficiency Syndromes in Children and Young Adults.

Replication repair deficiency (RRD) is a pan-cancer mechanism characterized by abnormalities in the DNA mismatch repair (MMR) system due to pathogenic variants in the PMS2, MSH6, MSH2, or MLH1 genes, and/or in the polymerase-proofreading genes POLE and POLD1. RRD predisposition syndromes (constitutional MMR deficiency, Lynch, and polymerase proofreading-associated polyposis) share overlapping phenotypic and biological characteristics. Moreover, cancers stemming from germline defects of one mechanism can acquire somatic defects in another, leading to complete RRD. Here we describe the recent advances in the diagnostics, surveillance, and clinical management for children with RRD syndromes. For patients with constitutional MMR deficiency, new data combining clinical insights and cancer genomics have revealed genotype-phenotype associations and helped in the development of novel functional assays, diagnostic guidelines, and surveillance recommendations. Recognition of non-gastrointestinal/genitourinary malignancies, particularly aggressive brain tumors, in select children with Lynch and polymerase proofreading-associated polyposis syndromes harboring an RRD biology have led to new management considerations. Additionally, universal hypermutation and microsatellite instability have allowed immunotherapy to be a paradigm shift in the treatment of RRD cancers independent of their germline etiology. These advances have also stimulated a need for expert recommendations about genetic counseling for these patients and their families. Future collaborative work will focus on newer technologies such as quantitative measurement of circulating tumor DNA and functional genomics to tailor surveillance and clinical care, improving immune surveillance; develop prevention strategies; and deliver these novel discoveries to resource-limited settings to maximize benefits for patients globally.

Variants in SART3 cause a spliceosomopathy characterised by failure of testis development and neuronal defects.

Squamous cell carcinoma antigen recognized by T cells 3 (SART3) is an RNA-binding protein with numerous biological functions including recycling small nuclear RNAs to the spliceosome. Here, we identify recessive variants in SART3 in nine individuals presenting with intellectual disability, global developmental delay and a subset of brain anomalies, together with gonadal dysgenesis in 46,XY individuals. Knockdown of the Drosophila orthologue of SART3 reveals a conserved role in testicular and neuronal development. Human induced pluripotent stem cells carrying patient variants in SART3 show disruption to multiple signalling pathways, upregulation of spliceosome components and demonstrate aberrant gonadal and neuronal differentiation in vitro. Collectively, these findings suggest that bi-allelic SART3 variants underlie a spliceosomopathy which we tentatively propose be termed INDYGON syndrome (Intellectual disability, Neurodevelopmental defects and Developmental delay with 46,XY GONadal dysgenesis). Our findings will enable additional diagnoses and improved outcomes for individuals born with this condition.

Early Infiltration of Innate Immune Cells to the Liver Depletes HNF4α and Promotes Extrahepatic Carcinogenesis.

Multiple studies have identified metabolic changes within the tumor and its microenvironment during carcinogenesis. Yet, the mechanisms by which tumors affect the host metabolism are unclear. We find that systemic inflammation induced by cancer leads to liver infiltration of myeloid cells at early extrahepatic carcinogenesis. The infiltrating immune cells via IL6-pSTAT3 immune-hepatocyte cross-talk cause the depletion of a master metabolic regulator, HNF4α, consequently leading to systemic metabolic changes that promote breast and pancreatic cancer proliferation and a worse outcome. Preserving HNF4α levels maintains liver metabolism and restricts carcinogenesis. Standard liver biochemical tests can identify early metabolic changes and predict patients' outcomes and weight loss. Thus, the tumor induces early metabolic changes in its macroenvironment with diagnostic and potentially therapeutic implications for the host. SIGNIFICANCE: Cancer growth requires a permanent nutrient supply starting from early disease stages. We find that the tumor extends its effect to the host's liver to obtain nutrients and rewires the systemic and tissue-specific metabolism early during carcinogenesis. Preserving liver metabolism restricts tumor growth and improves cancer outcomes. This article is highlighted in the In This Issue feature, p. 1501.

A rare genotype of biallelic mosaic variants in BCOR gene causing a bilateral ocular anterior segment dysgenesis and cataracts.

Oculofaciocardiodental (OFCD) syndrome is a rare X-linked dominant syndrome characterized by the involvement of the eyes, face, teeth, and heart with variable expressivity. The syndrome is caused by loss-of-function variants in the BCOR gene located on the X chromosome. OFCD affects only females with presumed embryonic lethality among males. We report a first case of a female with biallelic mosaic variants in BCOR gene, leading to a severe ocular phenotype including anterior segment dysgenesis, cataracts, and retinal involvement. The unique condition of biallelic mosaic loss-of-function mutations leads to a variable expression of an allele with the pathogenic variant, independent of the X-Inactivation pattern. This novel mechanism of co-existent biallelic mosaicism should be suspected in unexplained severe cases of OFCD.

The Genetic Landscape of Ischemic Stroke in Children - Current Knowledge and Future Perspectives.

Stroke in childhood has multiple etiologies, which are mostly distinct from those in adults. Genetic discoveries over the last decade pointed to monogenic disorders as a rare but significant cause of ischemic stroke in children and young adults, including small vessel and arterial ischemic stroke. These discoveries contributed to the understanding that stroke in children may be a sign of an underlying genetic disease. The identification of these diseases requires a detailed medical and family history collection, a careful clinical evaluation for the detection of systemic symptoms and signs, and neuroimaging assessment. Establishing an accurate etiological diagnosis and understanding the genetic risk factors for stroke are essential steps to decipher the underlying mechanisms, optimize the design of tailored prevention strategies, and facilitate the identification of novel therapeutic targets in some cases. Despite the increasing recognition of monogenic causes of stroke, genetic disorders remain understudied and therefore under-recognized in children with stroke. Increased awareness among healthcare providers is essential to facilitate accurate diagnosis in a timely manner. In this review, we provide a summary of the main single-gene disorders which may present as ischemic stroke in childhood and describe their clinical manifestations. We provide a set of practical suggestions for the diagnostic work up of these uncommon causes of stroke, based upon the stroke subtype and imaging characteristics that may suggest a monogenic diagnosis of ischemic stroke in children. Current hurdles in the genetic analyses of children with ischemic stroke as well as future prospectives are discussed.

Large scale population screening for Duchenne muscular dystrophy-Predictable and unpredictable challenges.

OBJECTIVE: Large deletions and duplications account for 65%-80% of pathogenic Duchenne muscular dystrophy (DMD) variants. A nationwide carrier screening for DMD was initiated in Israel in 2020. We assessed the carrier rate and spectrum of variants detected in a cohort of women screened for DMD carrier status and analyzed screening efficacy and challenges related to DMD population screening. METHODS: A cohort of 12,362 women were tested at a single institute using multiplex ligation-dependent probe amplification based copy number analysis of the 79 DMD exons. Consecutive sequencing of the primer region was performed when a single exon deletion was suspected. RESULTS: Deletions involving multiple exons were detected in seven cases and duplications involving multiple exons were found in four. Of these, nine were pathogenic based on previous reports and familial segregation testing, translating to a carrier rate of 1:1374. A family history was reported in three cases. Single exon deletions were suspected in 81 cases; further sequencing detected a single nucleotide variant affecting probe hybridization. These cases clustered according to ethnic origin. DISCUSSION: Population screening for DMD has a significant yield. Most carriers did not report a family history of dystrophinopathies. Screening should be adjusted for methodological limitations. Some cases may require extensive genetic counseling and work-up.

Di-genic inheritance of germline POLE and PMS2 pathogenic variants causes a unique condition associated with pediatric cancer predisposition.

Polymerase proofreading-associated polyposis (PPAP) and Lynch syndrome, caused by mutated POLE and mismatch repair (MMR) genes, respectively, are associated with adult-onset cancer. PPAP and MMR-deficient tumors are both hypermutated, and each has a unique mutational signature. We describe a 4.5-year-old boy with multiple café au lait spots who presented with metastatic Sonic Hedgehog-activated medulloblastoma, with partial response to intensive chemotherapy and immunotherapy. The tumor showed microsatellite stability, loss of PMS2 nuclear expression, and an exceptionally high tumor mutational burden of 276 Mut/Mb. Germline molecular analysis revealed an inherited heterozygous pathogenic POLE variant and a de novo heterozygous PMS2 pathogenic variant. The tumor featured the MMR, POLE, and POLE+MMR mutational signatures. This is the first description of a di-genic condition, which we named "POL-LYNCH syndrome," manifested by an aggressive ultra-mutant pediatric medulloblastoma with a unique genomic signature.

Evidence for increased breakthrough rates of SARS-CoV-2 variants of concern in BNT162b2-mRNA-vaccinated individuals.

The BNT162b2 mRNA vaccine is highly effective against SARS-CoV-2. However, apprehension exists that variants of concern (VOCs) may evade vaccine protection, due to evidence of reduced neutralization of the VOCs B.1.1.7 and B.1.351 by vaccine sera in laboratory assays. We performed a matched cohort study to examine the distribution of VOCs in infections of BNT162b2 mRNA vaccinees from Clalit Health Services (Israel) using viral genomic sequencing, and hypothesized that if vaccine effectiveness against a VOC is reduced, its proportion among breakthrough cases would be higher than in unvaccinated controls. Analyzing 813 viral genome sequences from nasopharyngeal swabs, we showed that vaccinees who tested positive at least 7 days after the second dose were disproportionally infected with B.1.351, compared with controls. Those who tested positive between 2 weeks after the first dose and 6 days after the second dose were disproportionally infected by B.1.1.7. These findings suggest reduced vaccine effectiveness against both VOCs within particular time windows. Our results emphasize the importance of rigorously tracking viral variants, and of increasing vaccination to prevent the spread of VOCs.

Survival Benefit for Individuals With Constitutional Mismatch Repair Deficiency Undergoing Surveillance.

PURPOSE: Constitutional mismatch repair deficiency syndrome (CMMRD) is a lethal cancer predisposition syndrome characterized by early-onset synchronous and metachronous multiorgan tumors. We designed a surveillance protocol for early tumor detection in these individuals. PATIENTS AND METHODS: Data were collected from patients with confirmed CMMRD who were registered in the International Replication Repair Deficiency Consortium. Tumor spectrum, efficacy of the surveillance protocol, and malignant transformation of low-grade lesions were examined for the entire cohort. Survival outcomes were analyzed for patients followed prospectively from the time of surveillance implementation. RESULTS: A total of 193 malignant tumors in 110 patients were identified. Median age of first cancer diagnosis was 9.2 years (range: 1.7-39.5 years). For patients undergoing surveillance, all GI and other solid tumors, and 75% of brain cancers were detected asymptomatically. By contrast, only 16% of hematologic malignancies were detected asymptomatically (P < .001). Eighty-nine patients were followed prospectively and used for survival analysis. Five-year overall survival (OS) was 90% (95% CI, 78.6 to 100) and 50% (95% CI, 39.2 to 63.7) when cancer was detected asymptomatically and symptomatically, respectively (P = .001). Patient outcome measured by adherence to the surveillance protocol revealed 4-year OS of 79% (95% CI, 54.8 to 90.9) for patients undergoing full surveillance, 55% (95% CI, 28.5 to 74.5) for partial surveillance, and 15% (95% CI, 5.2 to 28.8) for those not under surveillance (P < .0001). Of the 64 low-grade tumors detected, the cumulative likelihood of transformation from low-to high-grade was 81% for GI cancers within 8 years and 100% for gliomas in 6 years. CONCLUSION: Surveillance and early cancer detection are associated with improved OS for individuals with CMMRD.

MHC Haplotyping of SARS-CoV-2 Patients: HLA Subtypes Are Not Associated with the Presence and Severity of COVID-19 in the Israeli Population.

HLA haplotypes were found to be associated with increased risk for viral infections or disease severity in various diseases, including SARS. Several genetic variants are associated with COVID-19 severity. Studies have proposed associations, based on a very small sample and a large number of tested HLA alleles, but no clear association between HLA and COVID-19 incidence or severity has been reported. We conducted a large-scale HLA analysis of Israeli individuals who tested positive for SARS-CoV-2 infection by PCR. Overall, 72,912 individuals with known HLA haplotypes were included in the study, of whom 6413 (8.8%) were found to have SARS-CoV-2 by PCR. A total of 20,937 subjects were of Ashkenazi origin (at least 2/4 grandparents). One hundred eighty-one patients (2.8% of the infected) were hospitalized due to the disease. None of the 66 most common HLA loci (within the five HLA subgroups: A, B, C, DQB1, DRB1) was found to be associated with SARS-CoV-2 infection or hospitalization in the general Israeli population. Similarly, no association was detected in the Ashkenazi Jewish subset. Moreover, no association was found between heterozygosity in any of the HLA loci and either infection or hospitalization. We conclude that HLA haplotypes are not a major risk/protecting factor among the Israeli population for SARS-CoV-2 infection or severity. Our results suggest that if any HLA association exists with the disease it is very weak, and of limited effect on the pandemic.

Deletion in COL4A2 is associated with a three-generation variable phenotype: from fetal to adult manifestations.

Genetic alterations in COL4A2 are less common than those of COL4A1 and their fetal phenotype has not been described to date. We describe a three-generation family with an intragenic deletion in COL4A2 associated with a prenatal diagnosis of recurrent fetal intracerebral hemorrhage (ICH), and a myriad of cerebrovascular manifestations. Exome sequencing, co-segregation analysis, and imaging studies were conducted on eight family members including two fetuses with antenatal ICH. Histopathological evaluation was performed on the terminated fetuses. An intragenic heterozygous pathogenic in-frame deletion; COL4A2, c.4151_4168del, (p.Thr1384_Gly1389del) was identified in both fetuses, their father with hemiplegic cerebral palsy (CP), as well as other family members. Postmortem histopathological examination identified microscopic foci of heterotopias and polymicrogyria. The variant segregated in affected individuals demonstrating varying degrees of penetrance and a wide phenotypic spectrum including periventricular venous hemorrhagic infarction causing hemiplegic CP, polymicrogyria, leukoencephalopathy, and lacunar stroke. We present radiographic, pathological, and genetic evidence of prenatal ICH and show, for what we believe to be the first time, a human pathological proof of polymicrogyria and heterotopias in association with a COL4A2 disease-causing variant, while illustrating the variable phenotype and partial penetrance of this disease. We highlight the importance of genetic analysis in fetal ICH and hemiplegic CP.

Expanding the genotypic and phenotypic spectrum in a diverse cohort of 104 individuals with Wiedemann-Steiner syndrome.

Wiedemann-Steiner syndrome (WSS) is an autosomal dominant disorder caused by monoallelic variants in KMT2A and characterized by intellectual disability and hypertrichosis. We performed a retrospective, multicenter, observational study of 104 individuals with WSS from five continents to characterize the clinical and molecular spectrum of WSS in diverse populations, to identify physical features that may be more prevalent in White versus Black Indigenous People of Color individuals, to delineate genotype-phenotype correlations, to define developmental milestones, to describe the syndrome through adulthood, and to examine clinicians' differential diagnoses. Sixty-nine of the 82 variants (84%) observed in the study were not previously reported in the literature. Common clinical features identified in the cohort included: developmental delay or intellectual disability (97%), constipation (63.8%), failure to thrive (67.7%), feeding difficulties (66.3%), hypertrichosis cubiti (57%), short stature (57.8%), and vertebral anomalies (46.9%). The median ages at walking and first words were 20 months and 18 months, respectively. Hypotonia was associated with loss of function (LoF) variants, and seizures were associated with non-LoF variants. This study identifies genotype-phenotype correlations as well as race-facial feature associations in an ethnically diverse cohort, and accurately defines developmental trajectories, medical comorbidities, and long-term outcomes in individuals with WSS.

The effect of polyhydramnios degree on chromosomal microarray results: a retrospective cohort analysis of 742 singleton pregnancies.

PURPOSE: To analyze the risk for clinically significant microarray aberrations in pregnancies with polyhydramnios. METHODS: Data from all chromosomal microarray analyses (CMA) performed due to polyhydramnios between January 2013 and December 2019 were retrospectively obtained from the Ministry of Health Database. The rate of clinically significant (pathogenic and likely pathogenic) CMA findings in isolated and non-isolated polyhydramnios cohorts was compared to a local control group of 5541 fetuses with normal ultrasound, in which 78 (1.4%) abnormal results were demonstrated. Subgroup analyses were performed by the degree of polyhydramnios, week of diagnosis, maternal age, and the presence of additional sonographic anomalies. RESULTS: In the isolated polyhydramnios cohort, 19/623 (3.1%) clinically significant CMA aberrations were noted, a significantly higher rate compared to the control population. However, the risk for abnormal CMA results in the 158 cases with mild polyhydramnios (AFI 25-29.9, or maximal vertical pocket 8-11.9 cm) did not significantly differ from pregnancies with normal ultrasound. Of 119 cases of non-isolated polyhydramnios (most frequently associated with cardiovascular (26.1%) and brain (15.1%) anomalies), 8 (6.7%) abnormal CMA findings were noted, mainly karyotype-detectable. CONCLUSION: Mild polyhydramnios was not associated with an increased rate of clinically significant microarray results, compared to pregnancies with normal ultrasound. An extensive anatomical sonographic survey should be performed in pregnancies with polyhydramnios, with consideration of fetal echocardiography.

Monogenic Causes of Apparently Idiopathic Perinatal Intracranial Hemorrhage.

OBJECTIVE: Perinatal intracranial hemorrhage (pICH) is a rare event that occurs during the fetal/neonatal period with potentially devastating neurological outcome. However, the etiology of pICH is frequently hard to depict. We investigated the role of rare genetic variations in unexplained cases of pICH. METHODS: We performed whole-exome sequencing (WES) in fetuses and term neonates with otherwise unexplained pICH and their parents. Variant causality was determined according to the American College of Medical Genetics and Genomics (ACMG) criteria, consistency between suggested genes and phenotypes, and mode of inheritance. RESULTS: Twenty-six probands (25 families) were included in the study (9 with a prenatal diagnosis and 17 with a postnatal diagnosis). Intraventricular hemorrhage (IVH) was the most common type of hemorrhage (n = 16, 62%), followed by subpial (n = 4, 15%), subdural (n = 4, 15%), and parenchymal (n = 2, 8%) hemorrhage. Causative/likely causative variants were found in 4 subjects from 3 of the 25 families (12%) involving genes related to the brain microenvironment (COL4A1, COL4A2, and TREX-1). Additionally, potentially causative variants were detected in genes related to coagulation (GP1BA, F11, Von Willebrand factor [VWF], FGA, and F7; n = 4, 16%). A potential candidate gene for phenotypic expansion related to microtubular function (DNAH5) was identified in 1 case (4%). Fifty-five percent of the variants were inherited from an asymptomatic parent. Overall, these findings showed a monogenic cause for pICH in 12% to 32% of the families. INTERPRETATION: Our findings reveal a clinically significant diagnostic yield of WES in apparently idiopathic pICH and support the use of WES in the evaluation of these cases. ANN NEUROL 2021;89:813-822.

Ethnicity has a multiplex impact upon the risk of a full mutation expansion among female heterozygotes for FMR1 premutation.

PURPOSE: To evaluate whether ethnicity affects the risk of full mutation expansion among females heterozygous for FMR1 premutation. METHODS: Women who carry the FMR1 premutation alelle of Jewish origin who underwent fragile X prenatal diagnosis between 2011 and 2018 in two medical centers in Israel were included. The heterozygote women and fetuses were analyzed for the number of CGG repeats and AGG interruptions. RESULTS: Seven hundred sixty-six subjects were included. Parental ethnicity was fully concordant in 592 cases (Jewish, Ashkenazi, and non-Ashkenazi). Ashkenazi compared with non-Ashkenazi heterozygotes have a significantly higher mean number of CGG repeats (68 ± 8.7, 64 ± 6.4 respectively, P = 0.03) and a lower mean number of AGG interruptions (0.89 ± 0.83, 1.60 ± 1.18 respectively, p = 0.0001). Overall, 56/198 (28.2%) fetuses of Ashkenazi heterozygotes had an expansion to a full mutation compared with 6/98 among the non-Ashkenazi (6.1%) (p = 0.001). Multivariate analysis demonstrated that, in addition to CGG repeats and AGG interruptions (which contributed 68.3% of variance), ethnicity is an independent risk factor for a full mutation expansion (odds ratio [OR] = 2.04, p < 0.001) and accounted for 9% of the variation of a full mutation expansion. CONCLUSION: Apart from significant differences regarding the number of CGG repeats and AGG interruptions between Ashkenazi and non-Ashkenazi heterozygotes, ethnicity independently affects the risk of a full mutation.

Improving renal phenotype and evolving extra-renal features of 17q12 deletion encompassing the HNF1B gene.

BACKGROUND: HNF1B deletion/intragenic mutations are the most commonly identified genetic cause of congenital anomalies of the kidney and urinary tract (CAKUT) suggested by fetal ultrasound findings such as: parenchymal hyperechogenicity, overt cystic changes or gross morphological urinary system (UT) abnormalities. The postnatal evolution of these 17q12 deletions encompassing the HNF1B gene-associated findings has not been assessed in depth. METHODS: In this observational study, we present postnatal follow-up findings in 5 of 6 cases (one pregnancy was terminated on parental request) of fetal-onset cystic/hyperechogenic kidneys eventually diagnosed with 17q12 microdeletion encompassing the HNF1B gene between 2009 and 2017. RESULTS: Complete normalization of kidney parenchymal abnormalities and of depressed neonatal renal function was observed in 4/5 and 5/5 patients within 2-4.9 years and 1.5-8 months, respectively. All 5 patients had preserved normal renal function at 3-11 years of follow-up. The evolving later-onset renal features included: hypomagnesemia, hyperuricemia, urinary tract infection (UTI), and bilateral grade 3-4 vesicoureteral reflux and bladder diverticula in 3, 3, 2, and 1 patient, respectively. HNF1B gene deletion-associated extra-renal manifestations with delayed presentation were global developmental delay/autistic spectrum disorder (ASD), rolandic-type seizures, overweight, and borderline fasting hyperglycemia observed in 1-2 patients each. Family history was positive for small-size or asymptomatic cystic kidneys with normal function, diabetes mellitus, seizures, and mental/psychiatric problems in 3/6 cases. CONCLUSIONS: Fetal-onset HNF1B deletion-associated kidneys' parenchymal abnormalities confirmed postnatally with initially depressed renal function might undergo complete resolution within several years and few months, respectively. However, later-onset urinary tract, metabolic, and neurodevelopmental features of this mutation might appear over years. Therefore, genetic molecular evaluation/diagnosis and continuous follow-up for evolving features are mandatory in affected children.

Chromosomal microarray should be performed for cases of fetal short long bones detected prenatally.

PURPOSE: To investigate the prevalence of pathogenic and likely-pathogenic variants detected by chromosomal microarray analysis (CMA), among pregnancies with fetal short long bones diagnosed by ultrasound. METHODS: The study cohort was based on cases of chromosomal microarray analyses performed nationwide for the indication of short long bones. RESULTS: CMA was performed in 66 cases of short long bones. There were 4 cases with a pathogenic/likely pathogenic result (6%). The rate of chromosomal abnormalities was significantly higher compared to the background risk for copy number variations (CNVs) in pregnancies with no sonographic anomalies (P < 0.001). The yield of CMA in our cohort was significantly higher for both isolated and non-isolated cases, for cases in which the lowest estimated bone length percentile was above the 3rd percentile (below 5th percentile), and for cases diagnosed with short long bones after 22 weeks but not for cases diagnosed after 24 weeks. CONCLUSION: The yield of CMA in cases with short long bones (both isolated and non-isolated) is significantly higher than the background risk for chromosomal anomalies in pregnancies with no sonographic anomalies. This suggests that CMA should be offered in pregnancies with a diagnosis of fetal short long bones.