Early Diagnosis of Werner's Syndrome Using Exome-Wide Sequencing in a Single, Atypical Patient

Abstract

Genetic diagnosis of inherited metabolic disease is conventionally achieved through syndrome recognition and targeted gene sequencing, but many patients receive no specific diagnosis. Next-generation sequencing allied to capture of expressed sequences from genomic DNA now offers a powerful new diagnostic approach. Barriers to routine diagnostic use include cost, and the complexity of interpreting results arising from simultaneous identification of large numbers of variants. We applied exome-wide sequencing to an individual, 16-year-old daughter of consanguineous parents with a novel syndrome of short stature, severe insulin resistance, ptosis, and microcephaly. Pulldown of expressed sequences from genomic DNA followed by massively parallel sequencing was undertaken. Single nucleotide variants were called using SAMtools prior to filtering based on sequence quality and existence in control genomes and exomes. Of 485 genetic variants predicted to alter protein sequence and absent from control data, 24 were homozygous in the patient. One mutation - the p.Arg732X mutation in the WRN gene - has previously been reported in Werner's syndrome (WS). On re-evaluation of the patient several early features of WS were detected including loss of fat from the extremities and frontal hair thinning. Lymphoblastoid cells from the proband exhibited a defective decatenation checkpoint, consistent with loss of WRN activity. We have thus diagnosed WS some 15 years earlier than average, permitting aggressive prophylactic therapy and screening for WS complications, illustrating the potential of exome-wide sequencing to achieve early diagnosis and change management of rare autosomal recessive disease, even in individual patients of consanguineous parentage with apparently novel syndromes.

Keywords: WRN; Werner's syndrome; diabetes; insulin resistance; whole exome sequencing.

Figure 1

Early clinical features of Werner's syndrome in a 16-year-old female patient from a consanguineous family. (A) Clinical images of proband at (I) 7 years old and (II, III) 16 years old, showing truncal obesity and acanthosis nigricans in the neck and flexures at both ages, and marked loss of distal subcutaneous tissue from the arms by 16 years. (B) Growth chart showing failure of pubertal growth spurt, typical of Werner's syndrome. (C) Pedigree diagram (HOCM, hypertrophic obstructive cardiomyopathy; DM, type II diabetes mellitus).

Figure 2

Data analysis algorithm for whole exome sequencing data and location of pathogenic WRN mutation. Data analysis algorithm used for filtering all single nucleotide variants (SNVs) identified using exome-wide sequencing, with numbers of variants left at each filtering step.

Figure 3

Identification of a loss-of-function mutation in the WRN helicase (A). Homozygous p.Arg732X mutation in the WRN gene found in the proband, leading to truncation of the RecQ helicase domain and loss of both RecQ C-terminal (RQC) and helicase RNAseD C-terminal (HRDC) domains which mediate interaction with DNA and proteins by the WRN protein. (B) Impairment of the DNA decatenation checkpoint in lymphoblastoid cells from the proband. The inset shows a representative pseudo-mitosis. WT, wild type; Unt, untreated; WRN (R368X), cells from a Werner's syndrome patient homozygous for the p.Arg368X pathogenic variant.

Figure 4

(A) ATR protein expression in EBV-transformed lymphoblastoid cells from the proband compared to a wildtype control (WT). MCM2 is shown as a loading control. (B) Preserved G2-M cell cycle checkpoint in response to either ultraviolet light (UV) or ionizing radiation (IR), assessed by the rate of mitosis compared to untreated cells (Unt). Cells from a patient with biallelic hypomorphic mutation of ATR (ATR-S) are shown as a positive control for ATR dysfunction.