From pandemic to endemic: Divergence of COVID-19 positive-tests and hospitalization numbers from SARS-CoV-2 RNA levels in wastewater of Rochester, Minnesota.

Traditionally, public health surveillance relied on individual-level data but recently wastewater-based epidemiology (WBE) for the detection of infectious diseases including COVID-19 became a valuable tool in the public health arsenal. Here, we use WBE to follow the course of the COVID-19 pandemic in Rochester, Minnesota (population 121,395 at the 2020 census), from February 2021 to December 2022. We monitored the impact of SARS-CoV-2 infections on public health by comparing three sets of data: quantitative measurements of viral RNA in wastewater as an unbiased reporter of virus level in the community, positive results of viral RNA or antigen tests from nasal swabs reflecting community reporting, and hospitalization data. From February 2021 to August 2022 viral RNA levels in wastewater were closely correlated with the oscillating course of COVID-19 case and hospitalization numbers. However, from September 2022 cases remained low and hospitalization numbers dropped, whereas viral RNA levels in wastewater continued to oscillate. The low reported cases may reflect virulence reduction combined with abated inclination to report, and the divergence of virus levels in wastewater from reported cases may reflect COVID-19 shifting from pandemic to endemic. WBE, which also detects asymptomatic infections, can provide an early warning of impending cases, and offers crucial insights during pandemic waves and in the transition to the endemic phase.

Evaluation of the analytical sensitivity of ACON and LumiraDx SARS-CoV-2 rapid antigen tests using samples with presumed Omicron variant.

BACKGROUND: Analytical sensitivity of 2 rapid antigen tests was evaluated for detection of presumed SARS-CoV-2 Omicron variants and earlier variants of concern. METHODS: A total of 152 SARS-CoV-2 RNA positive samples (N and ORF1ab positive but S gene negative) were tested for SARS-CoV-2 antigen by ACON lateral flow and LumiraDx fluorescence immunoassays. Sensitivity within 3 viral load ranges was compared among these 152 samples and 194 similarly characterized samples collected prior to the circulation of the Delta variant (pre-Delta). RESULTS: Antigen was detected in >95% of pre-Delta and presumed Omicron samples for both tests at viral loads >500,000 copies/mL, and 65 to 85% of samples with 50,000-500,000 copies/mL. At viral load <50,000 copies/mL, antigen tests showed better sensitivity in detecting pre-Delta compared to Omicron variants. LumiraDx was more sensitive than ACON at low viral load. CONCLUSIONS: Antigen tests had decreased sensitivity for detecting presumed Omicron compared to pre-Delta variants at low viral load.

Neuropathological findings in COVID-19: an autopsy cohort.

The literature regarding the neuropathological findings in cases of SARS-CoV-2 infection, which causes coronavirus disease 2019 (COVID-19), is expanding. We identified 72 patients who died of COVID-19 (n = 48) or had recovered shortly before death (n = 24) and had autopsies performed at our institution (49 males, 23 females; median age at death 76.4 years, range: 0.0-95.0 years). Droplet digital polymerase chain reaction (ddPCR) for the detection of SARS-CoV-2 was performed (n = 58) in multiple brain regions. In cases the assay was successfully completed (n = 50), 98.0% were negative (n = 49) and 2% were indeterminate (n = 1). Most histologic findings were typical of the patient age demographic, such as neurodegenerative disease and arteriolosclerosis. A subset of cases demonstrated findings which may be associated with sequelae of critical illness. We identified 3 cases with destructive perivascular lesions with axonal injury, one of which also harbored perivascular demyelinating lesions. These rare cases may represent a parainfectious process versus sequelae of vascular injury. The lack of detectable SARS-CoV-2 by ddPCR or significant histologic evidence of direct infection suggests that active encephalitis is not a feature of COVID-19.

A reverse-transcription droplet digital PCR assay to detect and quantify SARS-CoV-2 RNA in upper respiratory tract specimens.

Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) is a positive-sense, single-stranded RNA virus that causes coronavirus disease 2019 (COVID-19). Symptoms are variable and range from asymptomatic or mild to severe (i.e., pneumonia) in both healthy and immunocompromised patients. We developed a reverse-transcription droplet digital PCR (RT-ddPCR) assay for quantification of SARS-CoV-2 RNA in clinical nasopharyngeal and oropharyngeal swab specimens and evaluated the assay, including reproducibility, agreement of results, analytical measurement range, linearity, analytical sensitivity, and analytical specificity. This quantitative assay had a LoD of 218 copies/mL of viral transport media, with a linear quantification range from 500 to 5,000,000 copies/mL (R2 of 0.9817 and 0.9853 for N1 and N2 targets, respectively). Qualitative agreement of categorical results was 90.5% (57/63) between the reference and RT-ddPCR assays. Quantitative agreement between the two assays showed correlation, with R2 of 0.9726 and 0.9713 for N1 and N2 targets, respectively. No cross-reactivity with common coronavirus strains was detected. This SARS-CoV-2 quantitative RT-ddPCR assay may be a useful tool for a variety of applications including identification of patients with low viral load and serial monitoring of viral load in respiratory tracts specimens of patients for evaluation of the efficacy of therapy for COVID-19.

SARS-CoV-2 RNA detection in Formalin-Fixed Paraffin-Embedded (FFPE) tissue by droplet digital PCR (ddPCR).

BACKGROUND: SARS-CoV-2 is an RNA virus that primarily causes respiratory disease; however, infection of other tissue has been reported. Evaluation of SARS-CoV-2 in tissue specimens may increase understanding of SARS-CoV-2 pathobiology. MATERIALS AND METHODS: A qualitative test for detection of SARS-CoV-2 in formalin-fixed paraffin-embedded (FFPE) tissues was developed and validated using droplet digital PCR (ddPCR), which has a lower limit of detection than reverse transcription (RT)-qPCR. After extraction of total RNA from unstained FFPE tissue, SARS-CoV-2 nucleocapsid (N1, N2) target sequences were amplified and quantified, along with human RPP30 as a control using the Bio-Rad SARS-CoV-2 ddPCR kit. RESULTS: SARS-CoV-2 was detected in all 21 known positive samples and none of the 16 negative samples. As few as approximately 5 viral copies were reliably detected. Since January 2021, many tissue types have been clinically tested. Of the 195 clinical specimens, the positivity rate was 35% with placenta and fetal tissue showing the highest percentage of positive cases. CONCLUSION: This sensitive FFPE-based assay has broad clinical utility with applications as diverse as pregnancy loss and evaluation of liver transplant rejection. This assay will aid in understanding atypical presentations of COVID-19 as well as long-term sequelae.

Comparison of In Situ Hybridization, Immunohistochemistry, and Reverse Transcription-Droplet Digital Polymerase Chain Reaction for Severe Acute Respiratory Syndrome Coronavirus 2 (SARS-CoV-2) Testing in Tissue.

CONTEXT.­: Small case series have evaluated severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) detection in formalin-fixed, paraffin-embedded tissue using reverse transcription-polymerase chain reaction, immunohistochemistry (IHC), and/or RNA in situ hybridization (RNAish). OBJECTIVE.­: To compare droplet digital polymerase chain reaction, IHC, and RNAish to detect SARS-CoV-2 in formalin-fixed, paraffin-embedded tissue in a large series of lung specimens from coronavirus disease 2019 (COVID-19) patients. DESIGN.­: Droplet digital polymerase chain reaction and RNAish used commercially available probes; IHC used clone 1A9. Twenty-six autopsies of COVID-19 patients with formalin-fixed, paraffin-embedded tissue blocks of 62 lung specimens, 22 heart specimens, 2 brain specimens, and 1 liver, and 1 umbilical cord were included. Control cases included 9 autopsy lungs from patients with other infections/inflammation and virus-infected tissue or cell lines. RESULTS.­: Droplet digital polymerase chain reaction had the highest sensitivity for SARS-CoV-2 (96%) when compared with IHC (31%) and RNAish (36%). All 3 tests had a specificity of 100%. Agreement between droplet digital polymerase chain reaction and IHC or RNAish was fair (κ = 0.23 and κ = 0.35, respectively). Agreement between IHC and in situ hybridization was substantial (κ = 0.75). Interobserver reliability was almost perfect for IHC (κ = 0.91) and fair to moderate for RNAish (κ = 0.38-0.59). Lung tissues from patients who died earlier after onset of symptoms revealed higher copy numbers by droplet digital polymerase chain reaction (P = .03, Pearson correlation = -0.65) and were more likely to be positive by RNAish (P = .02) than lungs from patients who died later. We identified SARS-CoV-2 in hyaline membranes, in pneumocytes, and rarely in respiratory epithelium. Droplet digital polymerase chain reaction showed low copy numbers in 7 autopsy hearts from ProteoGenex Inc. All other extrapulmonary tissues were negative. CONCLUSIONS.­: Droplet digital polymerase chain reaction was the most sensitive and highly specific test to identify SARS-CoV-2 in lung specimens from COVID-19 patients.

COVID-19-Associated Nonocclusive Fibrin Microthrombi in the Heart.

BACKGROUND: Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) and its resultant clinical presentation, coronavirus disease 2019 (COVID-19), is an emergent cause of mortality worldwide. Cardiac complications secondary to this infection are common; however, the underlying mechanisms of such remain unclear. A detailed cardiac evaluation of a series of individuals with COVID-19 undergoing postmortem evaluation is provided, with 4 aims: (1) describe the pathological spectrum of the myocardium; (2) compare with an alternate viral illness; (3) investigate angiotensin-converting enzyme 2 expression; and (4) provide the first description of the cardiac findings in patients with cleared infection. METHODS: Study cases were identified from institutional files and included COVID-19 (n=15: 12 active, 3 cleared), influenza A/B (n=6), and nonvirally mediated deaths (n=6). Salient information was abstracted from the medical record. Light microscopic findings were recorded. An angiotensin-converting enzyme 2 immunohistochemical H-score was compared across cases. Viral detection encompassed SARS-CoV-2 immunohistochemistry, ultrastructural examination, and droplet digital polymerase chain reaction. RESULTS: Male sex was more common in the COVID-19 group (P=0.05). Nonocclusive fibrin microthrombi (without ischemic injury) were identified in 16 cases (12 COVID-19, 2 influenza, and 2 controls) and were more common in the active COVID-19 cohort (P=0.006). Four active COVID-19 cases showed focal myocarditis, whereas 1 case of cleared COVID-19 showed extensive disease. Arteriolar angiotensin-converting enzyme 2 endothelial expression was lower in COVID-19 cases than in controls (P=0.004). Angiotensin-converting enzyme 2 myocardial expression did not differ by disease category, sex, age, or number of patient comorbidities (P=0.69, P=1.00, P=0.46, P=0.65, respectively). SARS-CoV-2 immunohistochemistry showed nonspecific staining, whereas ultrastructural examination and droplet digital polymerase chain reaction were negative for viral presence. Four patients (26.7%) with COVID-19 had underlying cardiac amyloidosis. Cases with cleared infection had variable presentations. CONCLUSIONS: This detailed histopathologic, immunohistochemical, ultrastructural, and molecular cardiac series showed no definitive evidence of direct myocardial infection. COVID-19 cases frequently have cardiac fibrin microthrombi, without universal acute ischemic injury. Moreover, myocarditis is present in 33.3% of patients with active and cleared COVID-19 but is usually limited in extent. Histological features of resolved infection are variable. Cardiac amyloidosis may be an additional risk factor for severe disease.

A Comparative Effectiveness Study of Newborn Screening Methods for Four Lysosomal Storage Disorders.

Newborn screening for one or more lysosomal disorders has been implemented in several US states, Japan and Taiwan by multiplexed enzyme assays using either tandem mass spectrometry or digital microfluidics. Another multiplex assay making use of immunocapture technology has also been proposed. To investigate the potential variability in performance of these analytical approaches, we implemented three high-throughput screening assays for the simultaneous screening for four lysosomal disorders: Fabry disease, Gaucher disease, mucopolysaccharidosis type I, and Pompe disease. These assays were tested in a prospective comparative effectiveness study using nearly 100,000 residual newborn dried blood spot specimens. In addition, 2nd tier enzyme assays and confirmatory molecular genetic testing were employed. Post-analytical interpretive tools were created using the software Collaborative Laboratory Integrated Reports (CLIR) to determine its ability to improve the performance of each assay vs. the traditional result interpretation based on analyte-specific reference ranges and cutoffs. This study showed that all three platforms have high sensitivity, and the application of CLIR tools markedly improves the performance of each platform while reducing the need for 2nd tier testing by 66% to 95%. Moreover, the addition of disease-specific biochemical 2nd tier tests ensures the lowest false positive rates and the highest positive predictive values for any platform.

Allelic spectrum of formiminotransferase-cyclodeaminase gene variants in individuals with formiminoglutamic aciduria.

BACKGROUND: Elevated plasma and urine formiminoglutamic acid (FIGLU) levels are commonly indicative of formiminoglutamic aciduria (OMIM #229100), a poorly understood autosomal recessive disorder of histidine and folate metabolism, resulting from formiminotransferase-cyclodeaminase (FTCD) deficiency, a bifunctional enzyme encoded by FTCD. METHODS: In order to further understanding about the molecular alterations that contribute to FIGLU-uria, we sequenced FTCD in 20 individuals with putative FTCD deficiency and varying laboratory findings, including increased FIGLU excretion. RESULTS: Individuals tested had biallelic loss-of-function variants in protein-coding regions of FTCD. The FTCD allelic spectrum comprised of 12 distinct variants including 5 missense alterations that replace conserved amino acid residues (c.223A>C, c.266A>G, c.319T>C, c.430G>A, c.514G>T), an in-frame deletion (c.1373_1375delTGG), with the remaining alterations predicted to affect mRNA processing/stability. These included two frameshift variants (c.990dup, c.1366dup) and four nonsense variants (c.337C>T, c.451A>T, c.763C>T, c.1607T>A). CONCLUSION: We observed additional FTCD alleles leading to urinary FIGLU elevations, and thus, providing molecular evidence of FTCD deficiency in cases identified by newborn screening or clinical biochemical genetic laboratory testing.

A Droplet Digital PCR Method for Severe Combined Immunodeficiency Newborn Screening.

Severe combined immunodeficiency (SCID) benefits from early intervention via hematopoietic cell transplantation to reverse T-cell lymphopenia (TCL). Newborn screening (NBS) programs use T-cell receptor excision circle (TREC) levels to detect SCID. Real-time quantitative PCR is often performed to quantify TRECs in dried blood spots (DBSs) for NBS. Yet, real-time quantitative PCR has inefficiencies necessitating normalization, repeat analyses, or standard curves. To address these issues, we developed a multiplex, droplet digital PCR (ddPCR) method for measuring absolute TREC amounts in one DBS punch. TREC and RPP30 levels were simultaneously measured with a Bio-Rad AutoDG and QX200 ddPCR system. DBSs from 610 presumed-normal, 29 lymphocyte-profiled, and 10 clinically diagnosed infants (1 X-linked SCID, 1 RAG1 Omenn syndrome, and other conditions) were tested. Control infants showed 14 to 474 TREC copies/µL blood. SCID infants, and other TCL conditions, had ≤15 TREC copies/µL. The ddPCR lower limit of quantitation was 14 TREC copies/µL, and the limit of detection was 4 TREC copies/µL. Intra-assay and interassay imprecision was <20% CV for DBSs at 54 to 60 TREC copies/µL. Testing 29 infants with known lymphocyte profiles resulted in a sensitivity of 88.9% and a specificity of 100% at TRECs <20 copies/µL. We developed a multiplex ddPCR method for the absolute quantitation of DBS TRECs that can detect SCID and other TCL conditions associated with absent or low TRECs and validated this method for NBS.

Evidence of genetic locus heterogeneity for familial bicuspid aortic valve.

OBJECTIVE: We sought to determine if the gene responsible for bicuspid aortic valve (BAV) in an extended family corresponds to previously reported loci for inherited forms of the disorder. BACKGROUND: Loci at 15q25.1-26 and 9q34 have been reported to be associated with cardiovascular abnormalities involving BAV. METHODS: Linkage analysis was performed on DNA collected from a large multigenerational family in which BAV disease segregates in an autosomal dominant manner, using microsatellite markers from the regions previously reported to segregate with the phenotype. RESULTS: Lod scores were determined for genetic markers near the NOTCH1 gene and for a locus on chromosome 15q25.1-26 previously reported as being linked to BAV. The lod scores were negative or less than 1.0 for all markers tested. CONCLUSIONS: There is no evidence of linkage of BAV in our pedigree to either the NOTCH1 gene or to the chromosome 15 locus. The disorder in this family appears to be caused by a gene at a novel locus.

Novel NOTCH1 mutations in patients with bicuspid aortic valve disease and thoracic aortic aneurysms.

OBJECTIVES: Bicuspid aortic valve is a common condition and is associated with a significantly increased risk of developing thoracic aortic aneurysms and acute aortic dissection. Patient-specific prediction of the risk of developing thoracic aortic aneurysm, however, is imprecise. We hypothesize that genotypic variations in patients with bicuspid aortic valves contribute to this observed variability in aortic phenotype. We, therefore, investigated the potential relationship between mutations in regions of NOTCH1 recently reported to be associated with bicuspid aortic valve and the phenotype of bicuspid aortic valve and thoracic aortic aneurysms in unrelated patients undergoing surgical repair. METHODS: We performed a targeted mutational analysis of NOTCH1 using genomic DNA from 48 unrelated subjects with concomitant bicuspid aortic valve and thoracic aortic aneurysm using denaturing high-performance liquid chromatography and DNA sequencing. We focused on exons in which mutations associated with bicuspid aortic valve have been reported previously. Results were compared with control subjects with trileaflet aortic valves (n = 94), bicuspid aortic valves, and normal aortas (n = 22) and in subjects with tricuspid aortic valves and thoracic aortic aneurysms (n = 28). RESULTS: Four unique, nonsynonymous (3 novel) variants were identified in 5 (10.4%) of 48 patients with concomitant bicuspid aortic valves and thoracic aortic aneurysms compared with only 3 (2.1%) of 144 control subjects (P = .02). Of these, 2 novel missense mutations, A1343V and P1390T, were observed only in patients with bicuspid aortic valves and tricuspid aortic aneurysms. CONCLUSIONS: This targeted analysis involving NOTCH1 exons previously implicated in familial and sporadic bicuspid aortic valve demonstrates overrepresentation of NOTCH1 missense variants among patients with bicuspid aortic valves and thoracic aortic aneurysms. Identification of aneurysm-predisposing susceptibility genes may lead to gene-directed surgical therapy of the ascending aorta for patients with bicuspid aortic valves.

Elevated expressions of osteopontin and tenascin C in ascending aortic aneurysms are associated with trileaflet aortic valves as compared with bicuspid aortic valves.

OBJECTIVE: Ascending aortic aneurysms (AscAAs) are a highly lethal condition whose pathobiology remains to be poorly understood. Although most AscAAs occur in the presence of a trileaflet aortic valve (TAV), a bicuspid aortic valve (BAV) is a common congenital anomaly associated with an increased risk for an AscAA and dissection independent of functional valve pathology but secondary to inherent structural abnormality of the aorta. The objective of this investigation was to compare the patterns of gene expression in aortas between TAV and BAV patients with the aim of identifying markers for AscAAs. METHODS: We used microarray analysis to first compare messenger RNA expressions between aneurysmal aortas from TAV patients (n=11) and those from BAV patients (n=11), identified genes overexpressed in TAV aneurysms, and compared expressions of the selected genes among TAV aneurysms, BAV aneurysms, and normal aortas (n=3). Finally, expressions of the selected genes were assessed by immunostaining of aortas from the TAV, BAV, and normal specimens. RESULTS: Two overexpressed genes in the TAV group, osteopontin (OPN) and tenascin C (TNC), were consistently more highly expressed in TAV aneurysms than in BAV aneurysms and normal aortas as determined by real-time reverse transcriptase quantitative polymerase chain reaction and immunohistochemistry. Differential staining revealed that OPN protein was concentrated in the medial smooth muscle and that TNC protein was concentrated around the vasa vasorum. CONCLUSIONS: We identified two novel potential markers, OPN and TNC, that are strongly associated with TAV aneurysms. The roles of OPN and TNC in influencing extracellular matrix remodeling in AscAAs warrant further investigation.

Bicuspid aortic valve and ascending aortic aneurysm are not associated with germline or somatic homeobox NKX2-5 gene polymorphism in 19 patients.

BACKGROUND: Bicuspid aortic valve is the most common congenital anomaly, occurring in 1% to 2% of the population. It is the most common reason for aortic valve replacement, and such individuals are at significantly increased risk of aortic complications. Despite the clinical significance of bicuspid aortic valve, its genetic basis remains unclear. The homeobox gene NKX2-5 occupies a central position in the hierarchy of cardiac determinants, and mutations in this gene are associated with bicuspid aortic valve in mice. We therefore investigated the presence of mutations in NKX2-5 among patients with bicuspid aortic valve and associated aneurysm. METHODS: Germline DNA was extracted from peripheral blood leukocytes and somatic DNA from diseased aortic tissues of 19 patients with bicuspid aortic valve and associated aortic aneurysm. Three patients with trileaflet aortic valve and aneurysm served as control subjects. The entire NKX2-5 coding sequence, including intron-exon boundaries, was screened for mutation by means of polymerase chain reaction, followed by DNA sequencing. RESULTS: Direct sequencing revealed a change in somatic (aortic) DNA 239A-->G, leading to synonymous amino acid alteration of Glu21Glu in one patient with bicuspid aortic valve and 1 control subject. There were no other alterations detected in the coding regions of germline or somatic genes. A known polymorphic change in the 3' untranslated region adjacent to exon 2 was detected in both bicuspid aortic valve and control samples. Discrepancies between germline and somatic DNA sequences were observed. CONCLUSION: Our study fails to demonstrate an association between bicuspid aortic valve and NKX2-5 mutation, as has been seen in mice. Our findings support the importance of sequencing somatic, as well as germline, DNA.

Molecular analysis of the spinal muscular atrophy and neuronal apoptosis inhibitory protein genes in Saudi patients with spinal muscular atrophy.

OBJECTIVE: Spinal muscular atrophy (SMA) is a common, often fatal, autosomal recessive disease leading to progressive muscle wasting and paralysis as a result of degeneration of anterior horn cells of the spinal cord. The prevalence of SMA cases in the Kingdom of Saudi Arabia (KSA) is much higher than the European and North American population. Deletions or mutations in 2 genes, telomeric form of the survival motor neuron (SMN1) and the neuronal apoptosis inhibitory protein (NAIP), are known to be associated with SMA. The aim of this study is to examine the deletions or interruptions of the SMN1 and NAIP genes in Saudi patients. METHODS: The study included 121 Saudi SMA patients [type I (60 patients); type II (26 patients); and type III (35 patients)]. The deletions or interruptions of the SMN1 and NAIP genes were detected by using polymerase chain reaction. The study was carried out at the King Fahad National Guard Hospital, Riyadh, KSA between 2000 and 2002. RESULTS: The homozygous deletions of exons 7 and 8 of the SMN1 gene were found in 94% and 87% of the patients. Exon 5 of the NAIP gene was deleted in 70%, but its deletion was more frequent in SMA type I (93%) as compared to type II (54%) and type III (43%). Seven patients with SMA diagnosis did not show any of the above homozygous deletions. All 230 control subjects had at least one copy of both SMN1 and NAIP genes, as expected. CONCLUSION: Our results demonstrate that the deletion rate (94%) of the SMN1 gene in Saudi SMA patients is similar, irrespective of types, compared with patients of other ethnic groups. We also show that the incidence of NAIP deletion is higher in the more severe SMA cases and the dual deletion of the SMN1 and NAIP genes are more common in Saudi SMA type I patients compared with patients of other ethnic groups.

Deletion mutations in the dystrophin gene of Saudi patients with Duchenne and Becker muscular dystrophy.

OBJECTIVE: The deletion in the dystrophin gene has been reported for many ethnic groups, but until now the mutations in this gene have not been thoroughly investigated in Saudi patients with Duchenne muscular dystrophy (DMD) and Becker muscular dystrophy (BMD). We examined the deletion pattern in the dystrophin gene of the Saudi patients applying multiplex-polymerase chain reaction (PCR). The aim of this study is to describe the outcome of our initial effort to identify mutations in the dystrophin gene in a representative group of Saudi patients with DMD and BMD. METHODS: Genomic deoxyribose nucleic acid was isolated from 41 patients with DMD and BMD (27 patients confirmed by muscle biopsy and 14 patients with clinical suspicion), 3 patients with limb girdle muscular dystrophy, 12 male relatives of the patients, and 5 healthy Saudi volunteers. A total of 25 exons around the deletion prone regions (hot spots) of the dystrophin gene were amplified. The study was carried out at the King Fahad National Guard Hospital, Riyadh, Kingdom of Saudi Arabia between 2000 and 2002. RESULTS: The deletion of one or more exons was found in 21 of 27 DMD and BMD patients confirmed by muscle biopsy. The deletion in the gene was detected in 5 of 14 patients with DMD diagnosis, but not confirmed by dystrophin staining of muscle biopsy. No deletion in the dystrophin gene was detected in control Saudi volunteers, the limb girdle dystrophy patients, and the relatives of patients, as expected. CONCLUSION: The present study suggests that intragenic dystrophin gene deletions occur with the same frequency in Saudi patients compared with other ethnic groups. The PCR-based deletion analysis provides a reasonable first step in the diagnostic care of Saudi patients who may be afflicted with DMD and BMD.