Down syndrome in diverse populations.

Down syndrome is the most common cause of cognitive impairment and presents clinically with universally recognizable signs and symptoms. In this study, we focus on exam findings and digital facial analysis technology in individuals with Down syndrome in diverse populations. Photos and clinical information were collected on 65 individuals from 13 countries, 56.9% were male and the average age was 6.6 years (range 1 month to 26 years; SD = 6.6 years). Subjective findings showed that clinical features were different across ethnicities (Africans, Asians, and Latin Americans), including brachycephaly, ear anomalies, clinodactyly, sandal gap, and abundant neck skin, which were all significantly less frequent in Africans (P < 0.001, P < 0.001, P < 0.001, P < 0.05, and P < 0.05, respectively). Evaluation using a digital facial analysis technology of a larger diverse cohort of newborns to adults (n = 129 cases; n = 132 controls) was able to diagnose Down syndrome with a sensitivity of 0.961, specificity of 0.924, and accuracy of 0.943. Only the angles at medial canthus and ala of the nose were common significant findings amongst different ethnicities (Caucasians, Africans, and Asians) when compared to ethnically matched controls. The Asian group had the least number of significant digital facial biometrics at 4, compared to Caucasians at 8 and Africans at 7. In conclusion, this study displays the wide variety of findings across different geographic populations in Down syndrome and demonstrates the accuracy and promise of digital facial analysis technology in the diagnosis of Down syndrome internationally. © 2016 Wiley Periodicals, Inc.

Next-generation sequencing using a pre-designed gene panel for the molecular diagnosis of congenital disorders in pediatric patients.

BACKGROUND: Next-generation sequencing (NGS) has revolutionized genetic research and offers enormous potential for clinical application. Sequencing the exome has the advantage of casting the net wide for all known coding regions while targeted gene panel sequencing provides enhanced sequencing depths and can be designed to avoid incidental findings in adult-onset conditions. A HaloPlex panel consisting of 180 genes within commonly altered chromosomal regions is available for use on both the Ion Personal Genome Machine (PGM) and MiSeq platforms to screen for causative mutations in these genes. METHODS: We used this Haloplex ICCG panel for targeted sequencing of 15 patients with clinical presentations indicative of an abnormality in one of the 180 genes. Sequencing runs were done using the Ion 318 Chips on the Ion Torrent PGM. Variants were filtered for known polymorphisms and analysis was done to identify possible disease-causing variants before validation by Sanger sequencing. When possible, segregation of variants with phenotype in family members was performed to ascertain the pathogenicity of the variant. RESULTS: More than 97% of the target bases were covered at >20×. There was an average of 9.6 novel variants per patient. Pathogenic mutations were identified in five genes for six patients, with two novel variants. There were another five likely pathogenic variants, some of which were unreported novel variants. CONCLUSIONS: In a cohort of 15 patients, we were able to identify a likely genetic etiology in six patients (40%). Another five patients had candidate variants for which further evaluation and segregation analysis are ongoing. Our results indicate that the HaloPlex ICCG panel is useful as a rapid, high-throughput and cost-effective screening tool for 170 of the 180 genes. There is low coverage for some regions in several genes which might have to be supplemented by Sanger sequencing. However, comparing the cost, ease of analysis, and shorter turnaround time, it is a good alternative to exome sequencing for patients whose features are suggestive of a genetic etiology involving one of the genes in the panel.

De novo trisomy 12p in twin girls with different levels of mosaicism.

We report on a pair of twins with trisomy 12p diagnosed postnatally. The girls were referred for dysmorphism and global developmental delay and have been followed from 10 months of age. They have different levels of mosaicism for both buccal cells and lymphocytes. Although their phenotypic features were similar, there were different degrees of severity which correlate with the different levels of mosaicism.

Rapid carrier screening for beta-thalassemia by single-step allele-specific PCR and detection.

OBJECTIVES: This study aimed to evaluate a rapid molecular carrier screening strategy for beta-thalassemia. DESIGN AND METHODS: Allele-specific PCR was combined with amplicon detection by dissociation curve analysis of SYBR Green I fluorescence in a single step. RESULTS: The presence of a particular mutation results in the amplification of a mutation-specific product and the dissociation temperature of each amplicon was highly reproducible. CONCLUSIONS: Homogeneous allele-specific PCR amplification and detection of multiple beta-globin mutations can serve as a rapid and inexpensive carrier screening tool.

Determining the cause of patchwork HBA1 and HBA2 genes: recurrent gene conversion or crossing over fixation events.

BACKGROUND AND OBJECTIVES: Recombinations are common between the two homologous alpha-globin genes. We report on the identification and characterization of two patchwork alpha-globin genes. DESIGN AND METHODS: Multiplex polymerase chain reaction assays were performed to rule out the presence of alpha-globin gene deletions and triplications. The HBA1 (alpha1-globin) and HBA2 (alpha2-globin) genes were individually amplified and sequenced. RESULTS: Two variants of the HBA1 and HBA2 genes were identified. One variant allele, alpha121, consists primarily of the HBA1 gene sequence except for a small segment of IVSII in which an octanucleotide segment has been replaced by an HBA2 -specific nucleotide. Conversely, the alpha212 variant consists primarily of the HBA2 gene sequence except for a segment of IVSII in which HBA2 -specific nucleotides at two sites have been replaced by HBA1-specific sequences. Both variant alleles are found in individuals of different ethnicity, geographical origin, and haplotype backgrounds. The simplest model for the origins of these patchwork alleles is a single crossover between a normal allele and an existing recombinant allele such as the -alpha(3.7) single gene deletion or the alphaalphaalpha(anti3.7) triplicated allele, but we cannot exclude a reciprocal double crossover or a non-reciprocal gene conversion between misaligned HBA1 and HBA2 genes. INTERPRETATION AND CONCLUSIONS: The a-globin patchwork alleles have arisen independently on several occasions, most likely through a single crossover between a normal and a recombinant allele. Further studies are necessary to evaluate the possible effect of these changes on alpha-globin gene expression.

De novo 3q22.1 q24 deletion associated with multiple congenital anomalies, growth retardation and intellectual disability.

We describe a boy with a de novo deletion of 15.67 Mb spanning 3q22.1q24. He has bilateral micropthalmia, ptosis, cleft palate, global developmental delay and brain, skeletal and cardiac abnormalities. In addition, he has bilateral inguinal hernia and his right kidney is absent. We compare his phenotype with seven other patients with overlapping and molecularly defined interstitial 3q deletions. This patient has some phenotypic features that are not shared by the other patients. More cases with smaller deletions defined by high resolution aCGH will enable better genotype-phenotype correlations and prioritizing of candidate genes for the identification of pathways and disease mechanisms.

FMR1 CGG repeat patterns and flanking haplotypes in three Asian populations and their relationship with repeat instability.

Hyper-expansion of a CGG repeat in the 5' untranslated region of the FMR1 gene followed by methylation and silencing is the predominant cause of Fragile X syndrome, the most common inherited mental retardation disorder. Most detailed studies of the FMR1 gene have focused on Caucasian populations and patients. We performed a detailed haplotype and linkage disequilibrium analysis of the FMR1 gene in a total of 454 unselected normal X chromosomes from three Asian populations, Chinese, Malay and Indian. Compared to Caucasians and African Americans, the diversity of normal FMR1 CGG repeat lengths, patterns and flanking haplotypes were lower in Asians. Strong linkage disequilibrium was observed between the CGG repeat and flanking FMR1 markers in all three Asian populations, with strong association between specific CGG repeat alleles and flanking marker alleles observed only in the Chinese and Malays. A test for randomness of distribution between FRAXA CGG repeat patterns and flanking FMR1 marker haplotypes also revealed a highly significant non-random distribution between CGG repeat patterns and flanking haplotypes in all three ethnic groups (P < 0.001). Extending previous findings in Caucasians and African Americans we present a novel statistical approach, using data from unselected population samples alone, to show an association between absence of at least one AGG interruption in any position (5', 3', or middle) and increased CGG repeat instability.

MS analysis of single-nucleotide differences in circulating nucleic acids: Application to noninvasive prenatal diagnosis.

The analysis of circulating nucleic acids has revealed applications in the noninvasive diagnosis, monitoring, and prognostication of many clinical conditions. Circulating fetal-specific sequences have been detected and constitute a fraction of the total DNA in maternal plasma. The diagnostic reliability of circulating DNA analysis depends on the fractional concentration of the targeted sequence, the analytical sensitivity, and the specificity. The robust discrimination of single-nucleotide differences between circulating DNA species is technically challenging and demands the adoption of highly sensitive and specific analytical systems. We have developed a method based on single-allele base extension reaction and MS, which allows for the reliable detection of fetal-specific alleles, including point mutations and single-nucleotide polymorphisms, in maternal plasma. The approach was applied to exclude the fetal inheritance of the four most common Southeast Asian beta-thalassemia mutations in at-risk pregnancies between weeks 7 and 21 of gestation. Fetal genotypes were correctly predicted in all cases studied. Fetal haplotype analysis based on a single-nucleotide polymorphism linked to the beta-globin locus, HBB, in maternal plasma also was achieved. Consequently, noninvasive prenatal diagnosis in a mother and father carrying identical beta-thalassemia mutations was accomplished. These advances will help in catalyzing the clinical applications of fetal nucleic acids in maternal plasma. This analytical approach also will have implications for many other applications of circulating nucleic acids in areas such as oncology and transplantation.