A metabolic signature for NADSYN1-dependent congenital NAD deficiency disorder.

Nicotinamide adenine dinucleotide (NAD) is essential for embryonic development. To date, biallelic loss-of-function variants in 3 genes encoding nonredundant enzymes of the NAD de novo synthesis pathway - KYNU, HAAO, and NADSYN1 - have been identified in humans with congenital malformations defined as congenital NAD deficiency disorder (CNDD). Here, we identified 13 further individuals with biallelic NADSYN1 variants predicted to be damaging, and phenotypes ranging from multiple severe malformations to the complete absence of malformation. Enzymatic assessment of variant deleteriousness in vitro revealed protein domain-specific perturbation, complemented by protein structure modeling in silico. We reproduced NADSYN1-dependent CNDD in mice and assessed various maternal NAD precursor supplementation strategies to prevent adverse pregnancy outcomes. While for Nadsyn1+/- mothers, any B3 vitamer was suitable to raise NAD, preventing embryo loss and malformation, Nadsyn1-/- mothers required supplementation with amidated NAD precursors (nicotinamide or nicotinamide mononucleotide) bypassing their metabolic block. The circulatory NAD metabolome in mice and humans before and after NAD precursor supplementation revealed a consistent metabolic signature with utility for patient identification. Our data collectively improve clinical diagnostics of NADSYN1-dependent CNDD, provide guidance for the therapeutic prevention of CNDD, and suggest an ongoing need to maintain NAD levels via amidated NAD precursor supplementation after birth.

Haploinsufficiency of SF3B2 causes craniofacial microsomia.

Craniofacial microsomia (CFM) is the second most common congenital facial anomaly, yet its genetic etiology remains unknown. We perform whole-exome or genome sequencing of 146 kindreds with sporadic (n = 138) or familial (n = 8) CFM, identifying a highly significant burden of loss of function variants in SF3B2 (P = 3.8 × 10-10), a component of the U2 small nuclear ribonucleoprotein complex, in probands. We describe twenty individuals from seven kindreds harboring de novo or transmitted haploinsufficient variants in SF3B2. Probands display mandibular hypoplasia, microtia, facial and preauricular tags, epibulbar dermoids, lateral oral clefts in addition to skeletal and cardiac abnormalities. Targeted morpholino knockdown of SF3B2 in Xenopus results in disruption of cranial neural crest precursor formation and subsequent craniofacial cartilage defects, supporting a link between spliceosome mutations and impaired neural crest development in congenital craniofacial disease. The results establish haploinsufficient variants in SF3B2 as the most prevalent genetic cause of CFM, explaining ~3% of sporadic and ~25% of familial cases.

The prevalence and molecular basis of hemoglobinopathies in Cambodia.

Blood counts, hemoglobin (Hb) high performance liquid chromatography (HPLC), and DNA analyses were performed on 260 children, aged 5 months to 16 years, at Siem Reap to assess the prevalence of thalassemia and other hemoglobinopathies in regional Cambodia. Hemoglobinopathies were present in 134 children (51.5%) with 20 abnormal genotypes identified. alpha-Thalassemia (thal) (35.4%) was the most prevalent disorder and the -alpha3.7 gene deletion was the most common alpha-globin gene abnormality. The - -SEA deletion and nondeletional forms of alpha-thal, Hb Constant Spring [Hb CS, alpha142, Term-->Gln, TAA-->CAA (alpha2)], Hb Paksé [alpha142, Term-->Tyr, TAA-->TAT (alpha2)] and triplicated alpha genes, were also present but at low frequencies. Hb E [beta26(B8)Glu-->Lys, GAG-->AAG] (28.8%) was the most common beta-globin gene abnormality, whilst beta-thal was only detected in two children (0.8% of cases). Although hemoglobinopathies were common, the majority of abnormalities detected (heterozygous -alpha3.7 and Hb E) were not clinically significant. On the basis of these findings, and with the majority of abnormalities being mild, it seems improbable that thalassemia represents a major health burden in this region of Cambodia.

Y-chromosome and mitochondrial DNA studies on the population structure of the Christmas Island community.

Christmas Island is a remote Australian territory located close to the main Indonesian island of Java. Y-chromosome and mitochondrial DNA (mtDNA) markers were used to investigate the genetic structure of the population, which comprises communities of mixed ethnic origin. Analysis of 12 Y-chromosome biallelic polymorphisms revealed a high level of gene diversity and haplotype frequencies that were consistent with source populations in southern China and Southeast Asia. mtDNA hypervariable segment I (HVS-I) sequences displayed high levels of haplotype diversity and nucleotide diversity that were comparable to various Asian populations. Genetic distances revealed extremely low mtDNA differentiation among Christmas Islanders and Asian populations. This was supported by the relatively high proportion of sequence types shared among these populations. The most common mtDNA haplogroups were M* and B, followed by D and F, which are prevalent in East/Southeast Asia. Christmas Islanders of European descent were characterized by the Eurasian haplogroup R*, and a limited degree of admixture was observed. In general, analysis of the genetic data indicated population affinities to southern Chinese (in particular from the Yunnan Province) and Southeast Asia (Thailand, Malaysia, and Cambodia), which was consistent with historical records of settlement. The combined use of these different marker systems provides a useful and appropriate model for the study of contemporary populations derived from different ethnic origins.

A standard protocol for single nucleotide primer extension in the human genome using matrix-assisted laser desorption/ionization time-of-flight mass spectrometry.

Analysis of single nucleotide polymorphisms (SNPs) has become an increasingly important area of research, with numerous applications in medical genetics, population genetics, forensic science, and agricultural biotechnology. Large-scale SNP analyses require the development of methodologies that are economical, flexible, accurate and capable of automation. Primer extension in conjunction with matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOFMS) is currently emerging as a potential method for high-throughput SNP genotyping. We have evaluated a number of published primer extension methods and refined a simple and robust protocol to analyze human autosomal disease-causing mutations and population genetic markers on the Y-chromosome. Twelve different variant sites were examined, and homozygotes, heterozygotes and hemizygotes were accurately typed. A 100% concordance was observed between SNP genotypes obtained using the MALDI-TOFMS technique and alternative genotyping methods, such as restriction fragment length polymorphism (RFLP) assays and denaturing high-performance liquid chromatography (DHPLC). Since multiple polymorphisms can be detected in single reactions, the method provides a cost-effective approach for SNP analysis. The protocol is also extremely flexible (able to accommodate new markers) and can be adapted to a number of platforms without the use of commercial kits.