KRT9 gene mutation as a reliable indicator in the prenatal molecular diagnosis of epidermolytic palmoplantar keratoderma.

Epidermolytic palmoplantar keratoderma (EPPK) is the most frequent form of such keratodermas. It is inherited in an autosomal dominant pattern and is clinically characterized by diffuse yellowish thickening of the skin on the palms and soles with erythematous borders during the first weeks or months after birth. EPPK is generally caused by mutations of the KRT9 gene. More than 26 KRT9 gene mutations responsible for EPPK have been described (Human Intermediate Filament Database, www.interfil.org), and many of these variants are located within the highly-conserved coil 1A region of the α-helical rod domain of keratin 9. Unfortunately, there is no satisfactory treatment for EPPK. Thus, prenatal molecular diagnosis or pre-pregnancy diagnosis is crucial and benefits those affected who seek healthy descendants. In the present study, we performed amniotic fluid-DNA-based prenatal testing for three at-risk pregnant EPPK women from three unrelated southern Chinese families who carried the KRT9 missense mutations p.Arg163Trp and p.Arg163Gln, and successfully helped two families to bear normal daughters. We suggest that before the successful application of preimplantation genetic diagnosis (PGD), and noninvasive prenatal diagnosis of EPPK that analyzes fetal cells or cell-free DNA in maternal blood, prenatal genetic diagnosis by amniocentesis or chorionic villus sampling (CVS) offers a quite acceptable option for EPPK couples-at-risk to avoid the birth of affected offspring, especially in low- and middle-income countries.

Genetic diagnosis of autosomal dominant polycystic kidney disease by targeted capture and next-generation sequencing: utility and limitations.

Mutation-based molecular diagnostics of autosomal dominant polycystic kidney disease (ADPKD) is complicated by genetic and allelic heterogeneity, large multi-exon genes, and duplication sequences of PKD1. Recently, targeted resequencing by pooling long-range polymerase chain reaction (LR-PCR) amplicons has been used in the identification of mutations in ADPKD. Despite its high sensitivity, specificity and accuracy, LR-PCR is still complicated. We performed whole-exome sequencing on two unrelated typical Chinese ADPKD probands and evaluated the effectiveness of this approach compared with Sanger sequencing. Meanwhile, we performed targeted gene and next-generation sequencing (targeted DNA-HiSeq) on 8 individuals (1 patient from one family, 5 patients and 2 normal individuals from another family). Both whole-exome sequencing and targeted DNA-HiSeq confirmed c.11364delC (p.H3788QfsX37) within the unduplicated region of PKD1 in one proband; in the other family, targeted DNA-HiSeq identified a small insertion, c.401_402insG (p.V134VfsX79), in PKD2. These methods do not overcome the screening complexity of homology. However, the true positives of variants confirmed by targeted gene and next-generation sequencing were 69.4%, 50% and 100% without a false positive in the whole coding region and the duplicated and unduplicated regions, which indicated that the screening accuracy of PKD1 and PKD2 can be largely improved by using a greater sequencing depth and elaborate design of the capture probe.

RET proto-oncogene genetic screening of families with multiple endocrine neoplasia type 2 optimizes diagnostic and clinical management in China.

BACKGROUND: Genetic screening for germline mutations in the RET proto-oncogene has been extensively exploited worldwide to optimize the diagnostic and clinical management of multiple endocrine neoplasia type 2 (MEN2) patients and their relatives. However, a distinct lag period exists not only in the recognition but also in the medical treatment of patients with MEN2. Here we present a comprehensive genetic and clinical analysis of MEN2 among Chinese families followed from 1975 to 2011. Our series comprises 36 index cases and 134 relatives from 11 independent families. METHODS: Genetic diagnosis was performed in all participants by direct sequencing all relevant RET exons. Thyroidectomy was performed in 50 patients with varying cervical neck dissection procedures. Patients with pheochromocytoma (PHEO) underwent specific surgery. Demographic, clinical profiles, mutation types, tumor histopathologic features, and follow-up records were systematically analyzed. RESULTS: The RET mutations p.C634Y (n=34), p.C634R (n=6), p.C618S (n=13), p.V292M/R67H/R982C (n=7), p.L790F (n=2), and p.C634Y/V292M/R67H/R982C (n=1) were confirmed in 31 index cases and then identified in 32 at-risk relatives (mutation carriers), with MEN2A as the most common clinical subtype. The overall penetrance of PHEO in patients with MEN2A was 46.7%. A total of 50 patients underwent thyroidectomy, and there was a significant lowering of their mean age at thyroidectomy and the tumor diameter of the mutation carriers that were detected and operated on compared with the index cases (age at first surgery: 29.3 vs. 39.3 years, p<0.05; maximum size: 1.1 vs. 3.3 cm, p<0.001). There was also a decrease in the TNM staging and the proportion of patients who underwent inappropriate initial thyroid surgery (pN1: 31.6% vs. 100%, p<0.001; inappropriate surgery: 0% vs. 29%). Meanwhile, disease-free survival (DFS) increased (DFS: 100% vs. 58.1%, p<0.05). Both medullary thyroid carcinoma-specific (n=1) and PHEO-specific (n=5) deaths were reported during the study period. CONCLUSIONS: Our results further substantiate that gene scanning of all relevant RET exons is a powerful tool in the management of MEN2 patients, especially in asymptomatic carriers, and has led to earlier diagnosis and more complete initial treatment of patients with MEN2 in China.