Werner Syndrome

    The clinical phenotype of WS (WS; MIM# 277700) has been succinctly summarized as a “caricature of aging”. WS patients usually develop normally until they reach the second decade of life.  The first clinical sign is a lack of the pubertal growth spurt during the teen years.  In their 20s and 30s, patients begin to suffer from skin atrophy, and loss and graying of hair.  The loss of fat is often associated with characteristic ulcerations around the elbows and ankles which eventually may require amputation. Other complications include type 2 diabetes mellitus; osteoporosis (especially of the lower limbs, in contrast to the preferential involvement of vertebrae in usual aging); bilateral ocular cataracts (requiring surgery at a median age of 30); gonadal atrophy (with early loss of fertility); premature and severe forms of arteriosclerosis (including atherosclerosis, arteriolosclerosis and medial calcinosis) and peripheral neuropathy. Multiple cancers have been observed by middle age; in contrast to usual aging, however, these include a disproportionate number of sarcomas. Our recent survey of WS patients with a molecularly confirmed diagnosis revealed that the prevalence of cataracts was 100% (87/87). The prevalence of osteoporosis was 91%, hypogonadism 80%, diabetes mellitus 71%, and atherosclerosis 40% at the time of diagnosis. Median age of death in the most recent study was 54 years, a significant increase over what had been observed several decades ago, perhaps the result of improved medical management. The most common cause of death was myocardial infarction.

Age 8             Age 21         Age 36                 Age 56

Figure 1. A Werner syndrome patient with homozygous null WRN mutations. Although apparently normal at ages 8, cataracts were diagnosed at age 36 and severe ankle ulcerations were recorded at age 56. (Registry# SANAN1010).

The gene (WRN) responsible for WS was identified at the University of Washington by positional cloning in 1996. Sequence analysis and subsequent biochemical studies revealed that the human WRN protein possesses both exonuclease and helicase functions.  We recently reported over 100 unique WRN mutations. 

Figure 2. WRN disease mutations in classical WS patients.  WRN mutations in WS patients.  A diagram of the full-length wild type WRN protein is shown, with its N-terminus on the left (N) and C-terminus on the right (C). Known functional domains are marked with darker shades; the exonuclease domain, the helicase domain, the RecQ helicase conserved region (RQC), the helicase RNaseD C-terminal conserved region (HDRC), and the nuclear localization signal (NLS). Mutations are grouped according to canonical classes and further identified by their amino acid changes. Splicing mutations are indicated by the affected exons.  Splicing mutations that result in identical exon skipping are combined and indicated by the number of unique mutations as in (2). Deep intron mutations that create new exons are indicated as “Ins” along with the flanking exons. Splice mutations that create a new splice site (“nss”) are indicated as such. Genomic rearrangements, either deletion (Del) or duplication (dup), are shown at corresponding protein locations, with regions extending beyond this figure in dotted lines. * indicates uncertainty in the interpretation of array CGH results

“Atypical” Werner Syndrome

    Atypical Werner syndrome (AWS) patients were initially referred to our International Registry for molecular diagnosis of Werner syndrome but had wild type WRN coding regions and normal levels and sizes of the WRN protein by Western blots. Such cases were categorized as having AWS. We identified novel missense mutations in LMNA  among our AWS cases (Fig. 3).


Figure 3. Atypical Werner syndrome patient with a heterozygous LMNA mutation. This 36 year-old Norwegian male carries a L140R mutation and has cataracts, osteoporosis and an aged-appearance.

    The LMNA gene encodes nuclear intermediate filaments, lamin A and lamin C. The mature lamin A molecule consists of a globular head domain, a-helical coiled coil domain, and globular tail domain. Within the a-helical coiled coil domain, there is a heptad repeat region that is thought to be involved in the molecular interaction. The locations of R133L and L140R at the surface of a heptad repeat suggested that the mutations might not affect the structure of the lamin A/C dimer itself, but rather might perturb intermolecular interactions. (Fig. 4).

    We also identified a heterozygous substitution of the last nucleotide of LMNA exon 11 (c.1968G>A, Q656Q) in 36 year-old male with progeroid features. Although this change does not change the amino acid, it leads to the weak activation of the same cryptic splicing site as in Hutchinson-Gilford progeria syndromes. HPGS is caused by the point mutation in exon 11 of LMNA, which generates a cryptic splicing site and causes 50-amino acid in-frame deletion. The accumulation of this deletion-mutant lamin A, termed progerin is thought be responsible for the phenotypic presentation of HGPS. The progerin is found in the senescent cells in culture or cells derived from the normal old individuals, suggesting the role of progerin in normal aging process. The lower level of progerin found in our patient corresponds to the late onset of the symptoms.  

Figure 4. LMNA mutations identified in AWS. The diagram shows the structure of lamin A with N-terminus on the left and C-terminus on the right. It consists of globular head domain, a-helical coiled coil domain and globular tail domain. Within a-helical coiled coil domain, 1A, 1B (containing heptad repeats), 2A 1B1 and 2B2 sections are connected by the flexible linkers. A NLS is located between coil domain and the globular tail domain. Heterozygous amino acid changes and a splicing mutation that results in the in-frame deletion (?50) in AWS patients are shown below the diagram.

    Currently, standard approache to identify the causal variant is the next generation sequencing, exome sequencingand, more recently, whole genome sequencing methodologies. Exome sequencing involves targeted sequencing of all protein-coding subsequences (the exome). This is feasible because the exome consists of only ~5% of the human genome. The effort to identify AWS through the exome sequencing is currently in progress with our collaborator, Dr. Deborah Nickerson, Department of Genome Sciences, University of Washington. A combination of next generation sequencing, SNP arrays and candidate gene sequencing have successfully identified novel mutations in subsets of AWS cases. Those AWS loci highlight major roles in DNA damage repair and response: LMNA (nuclear structure and chromatin interaction), POLD1 (DNA polymerase delta), SPRTN (recruitment of translesional DNA polymerase eta), ERCC4 (nucleotide excision repair), MDM2 (an inhibitor of p53), CTC1 (telomere replication), SMAD4 (TGF-beta pathway) and SAMHD1 (regulation of dNTP pools). Cases of a BSCL2 mutation responsible for Seip syndrome as well as mosaic trisomy 8 were also identified. These findings continue to support the concept of genomic instability as a major mechanism of biological aging. Biological materials derived from the patients and family members can be made available to investigators upon request.

Hutchinson Gilford Progeria Syndrome

    Hutchinson Gilford Progeria Syndrome is a rare genetic disease that accelerates the aging process to about seven times the normal rate. Because of this accelerated aging, a child of ten years will have similar respiratory, cardiovascular, and arthritic conditions that a 70-year-old would have. Children from all races and cultures from around the world have been affected. Some physical features of Progeria children include dwarfism, wrinkled/aged-looking skin, baldness, and a pinched nose. Mental growth is equivalent to other children of the same age. Most children with Progeria live no longer than their early teenage years, though one or two have lived to be as old as 20 or 21.

    In April 0f 2003, two groups of scientists have independently identified that a point mutation in the LMNA gene, which encode Lamin proteins. The mutation in the LMNA gene from patients suffering Hutchinson Gilford Progeria cause the LMNA mRNA to splice differently. Lamins are microfilaments in the nucleus, and is important in maintaining the proper structure of the nuclei. Mutations in LMNA were reported to cause many diseases referred to as "laminopathies". They include Emery-Drefuss muscular dystrophy (EDM2), dilated cardiomyopathy type 1A (CMD1A), limb-girdle muscular dystrophy type 1B (LGMD1B), familiar partial lipodystrophy (FPLD), Charcot-Marie-Tooth disease type 2 (CMT2B1), mandibuloacral dysplasia (MAD). Unlike Werner Syndrome mutation, the effect of the mutation seems to be dominant negative.

Population Genetic Study

    The laboratory participates in NLTCS project, which is a long term study on the welfare of the elderly population in the United States. The project intends to access and monitor the conditions of the elderly people over time, and the laboratory serves as tissue and DNA bank for these subjects and is responsible to provide genetic information on the subjects. We performed genotyping, sequencing, and/or dosage measurement on genes implicated in the process of aging, oxidative damage, DNA damage, and fat deposition. This information will be compiled into a database for further statistical analysis including family health history, daily activities, medical history, and current health conditions to evaluate various aspects of the activities of the elderly, and further influence the future US political policy on the elderly.

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