Diagnosis for choroideremia in a large Chinese pedigree by next­generation sequencing (NGS) and non­invasive prenatal testing (NIPT).

To develop an effective strategy to isolate and use cell­free fetal DNA (cffDNA) for the combined use of next­generation sequencing (NGS) for diagnosing choroideremia and non­invasive prenatal testing (NIPT) for Y chromosome determination, a large Chinese family with an X­linked recessive disease, choroideremia, was recruited. Cell­free DNA was extracted from maternal plasma, and SRY polymerase chain reaction amplification was performed using NIPT. Sanger sequencing was subsequently used for fetal amniotic fluid DNA verification. A nonsense mutation (c.C799T:p.R267X) of the CHM gene on the X chromosome of the proband (IV:7) and another 5 males with choroideremia were detected, while 3 female carriers with no symptoms were also identified. The fetus (VI:7) was identified as female from the cffDNA, and the same heterozygous nonsense mutation present in her mother was also confirmed. At one and a half years of age, the female baby did not present with any associated symptoms of choroideremia. Therefore, cffDNA was successfully used for the combined use of NGS for diagnosing choroideremia in a large Chinese pedigree, and NIPT for Y chromosome determination. This approach should result in a markedly increased use of prenatal diagnosis and improvement, and more sophisticated clinical management of diseases in China and other developing countries. The establishment of a highly accurate method for prenatal gene diagnosis will allow for more reliable gene diagnosis, improved genetic counseling, and personalized clinical management of our patients.

Identification of a novel heterozygous missense mutation in the CACNA1F gene in a chinese family with retinitis pigmentosa by next generation sequencing.

BACKGROUND: Retinitis pigmentosa (RP) is an inherited retinal degenerative disease, which is clinically and genetically heterogeneous, and the inheritance pattern is complex. In this study, we have intended to study the possible association of certain genes with X-linked RP (XLRP) in a Chinese family. METHODS: A Chinese family with RP was recruited, and a total of seven individuals were enrolled in this genetic study. Genomic DNA was isolated from peripheral leukocytes, and used for the next generation sequencing (NGS). RESULTS: The affected individual presented the clinical signs of XLRP. A heterozygous missense mutation (c.1555C>T, p.R519W) was identified by NGS in exon 13 of the CACNA1F gene on X chromosome, and was confirmed by Sanger sequencing. It showed perfect cosegregation with the disease in the family. The mutation at this position in the CACNA1F gene of RP was found novel by database searching. CONCLUSION: By using NGS, we have found a novel heterozygous missense mutation (c.1555C>T, p.R519W) in CACNA1F gene, which is probably associated with XLRP. The findings might provide new insights into the cause and diagnosis of RP, and have implications for genetic counseling and clinical management in this family.

Development of RAPD-SCAR markers for Lonicera japonica (Caprifolicaceae) variety authentication by improved RAPD and DNA cloning / Desarrollo de marcadores RAPD-SCAR para Lonicera japonica Thunb. (Caprifolicaceae) para la autenticación de variedades mediante RAPD mejorados y clonación de ADN

Genetic diversity within a species is a common feature, which plays a vital role in its survival and adaptability, and is important for the identification and authentication of a species. Lonicera japonica is a traditionally used medicinal plant, which have been recently genetically characterized by an improved ran- dom amplified polymorphic DNA (RAPD) analysis. In this study, the molecular markers on the basis of these RAPD fragments have been developed to identify specific L. japonica variety. The DNAs were extracted from fresh young leaves of different samples of L. japonica collected from Shenzhen, Yichang, Leshan, Emei and Loudi, China. The DNA materials were amplified using improved RAPD PCR. Different RAPD bands were excised, cloned and developed for stable sequence-characterized amplified region (SCAR) markers with differ- ent species. Two SCAR markers, JYH3-3 and JYH4-3, have been successfully cloned from improved RAPD fragments. The SCAR marker JYH3-3 was found specific for all of the L. japonica samples collected from the different regions, and another marker JYH 4-3 was strictly specific to the Shenzhen sample from Guangdong province, which is geographically distant from Hubei, Sichuan and Hunan Provinces (source of other L. japonica samples). The marker JYH3-3 was found as specific molecular marker for the identification of L. japonica, while JYH4-3 was found as molecular marker strictly specific for the Shenzhen sample. The developed SCAR mark- ers might serve as more specific molecular markers for L. japonica variety authentication. The combination of improved RAPD analysis and SCAR marker development have resulted useful tools to study the genetic variety of any organism, which we have successfully applied here in L. japonica.

La diversidad genética dentro de una especie es una característica común, que juega un papel vital en su supervivencia y adaptabilidad, y es importante para la identificación y la autenticación de una especie. Lonicera japonica es una planta medicinal utilizada tradicionalmente, que han sido recientemente caracterizada genéticamente por amplificación aleatoria mejorada de ADN polimórfico (RAPD). En este estudio, los marcadores moleculares basados en estos fragmentos de RAPD se han desarrollado para identificar una variedad específica de L. japonica. Los ADN se extrajeron de las hojas jóvenes frescas de diferentes muestras de L. japonica recogidas de Shenzhen, Yichang, Leshan, Emei y Loudi, China. Los materiales de ADN fueron amplificados utilizando el RAPD PCR mejorado. Diferentes bandas RAPD fueron extraídas, clonadas y desarrolladas para las regiones amplificadas de secuencia conocida (SCAR) con marcado- res de diferentes especies. Dos marcadores SCAR, JYH3-3 y JYH4-3, se clonaron con éxito de los RAPD mejorados. El marcador SCAR JYH3-3 se encontró específico para todas las muestras de L. japonica recolectadas en las diferentes regiones, mientras que el otro marcador JYH4-3 era estrictamente específico para la muestra de Shenzhen de la provincia de Guangdong, que está geográficamente distante de Hubei, Sichuan y Provincias Hunan (fuente de otras muestras de L. japonica). Se encontró que JYH3-3 es un marcador molecular específico para la identificación de L. japonica, mientras que JYH4-3 se encontró como marcador molecular estrictamente específico para la muestra de Shenzhen. Los marcadores SCAR desarrollados podrían servir como marcadores moleculares más específicos para la autenticación de la variedad L. japonica. La combi- nación de RAPD mejorado y el desarrollo del marcador SCAR han dado como resultado herramientas útiles para el estudio de la variedad genética de cualquier organismo, que hemos aplicado con éxito en L. japonica.

Development of RAPD-SCAR markers for Lonicera japonica (Caprifolicaceae) variety authentication by improved RAPD and DNA cloning.

Genetic diversity within a species is a common feature, which plays a vital role in its survival and adaptability, and is important for the identification and authentication of a species. Lonicera japonica is a traditionally used medicinal plant, which have been recently genetically characterized by an improved ran- dom amplified polymorphic DNA (RAPD) analysis. In this study, the molecular markers on the basis of these RAPD fragments have been developed to identify specific L. japonica variety. The DNAs were extracted from fresh young leaves of different samples of L. japonica collected from Shenzhen, Yichang, Leshan, Emei and Loudi, China. The DNA materials were amplified using improved RAPD PCR. Different RAPD bands were excised, cloned and developed for stable sequence-characterized amplified region (SCAR) markers with differ- ent species. Two SCAR markers, JYH3-3 and JYH4-3, have been successfully cloned from improved.RAPD fragments. The SCAR marker JYH3-3 was found specific for all of the L. japonica samples collected from the different regions, and another marker JYH 4-3 was strictly specific to the Shenzhen sample from Guangdong province, which is geographically distant from Hubei, Sichuan and Hunan Provinces (source of other L. japonica samples). The marker JYH3-3 was found as specific molecular marker for the identification of L. japonica, while JYH4-3 was found as molecular marker strictly specific for the Shenzhen sample. The developed SCAR markers might serve as more specific molecular markers for L. japonica variety authentication. The combination of improved RAPD analysis and SCAR marker development have resulted useful tools to study the genetic variety of any organism, which we have successfully applied here in L. japonica.