The proband's initial labwork at the time of consultation revealed a plasma TG level of 20.6 mmol/L with a high-density lipoprotein cholesterol (HDL-C) level of 0.56 mmol/L. Her fasting glucose and insulin levels on oral antidiabetic therapy but not insulin were 14.2 mmol/L and 68.1 mU/L (normal <10 mU/L), respectively, with a HbA1C of 11.2%. Her creatinine was elevated at 255 μmol/L with overt proteinuria of 2.68 g/24 hours. Her total alkaline phosphatase of 416 U/L (normal < 330 U/L) demonstrated increases in both the liver and bone isoenzymes. Abdominal ultrasound revealed fatty infiltration of the liver and splenic enlargement (14 cm in length). Both her electrocardiogram and echocardiogram were normal.

The results of lipoprotein analyses of the proband, proband's mother and two sisters are shown in Table 1. The proband demonstrated significant hypertriglyceridemia (TG = 20.6 mmol/L) and hypoalphalipoproteinemia (HDL-C = 0.56 mmol/L). Both the proband's mother and older sister displayed mildly elevated plasma TG whereas the younger sister tested had relatively normal plasma TG. Plasma low density lipoprotein-cholesterol (LDL-C) levels were higher among the mother and younger sister compared to the others. The proband demonstrated the lowest LDL-C and HDL-C levels of all four members of the kindred examined.

Treatment of the proband's hypertriglyceridemia required the use of multiple therapies, including a low-fat diet (< 20 g fat per day), insulin (glargine 14 units/day), pioglitazone (45 mg/day), atorvastatin (40 mg/day), and omega-3-acid ethylesters (Omacor) (2 g bid). As a result of therapy, TG levels decreased by ~84% to 3.30 mmol/L and HDL-C levels improved by ~252% to 1.41 mmol/L.

Informed consent was obtained from all available subjects or their guardians, and the study was approved by the institutional review board of the University of Western Ontario (#07920E). DNA and lipoprotein analyses were unavailable for the father, one sister (aged 18 years), and brother (aged 29 years). Genomic DNA from the four available study subjects was isolated from whole blood (Puregene, Gentra Systems, Minneapolis, MN). Exons 1 to 23 of ALMS1 were amplified using the primers in Table 2. The final volume of 50 μL contained 32 pmol of each primer, 0.2 mM each of dATP, dCTP, dGTP, and dTTP, 1.5 mM MgCl₂, 50 mM KCl, 20 mM Tris-HCl (pH 8.4), and 2.5 units of Taq platinum DNA polymerase (Life Technologies, Mississauga, Ontario, Canada). DNA amplifications were performed with denaturing at 94°C for 5 minutes, followed by 30 cycles of a denaturing step at 94°C, an annealing step at 60°C, and an extension step at 72°C, each for 30 seconds. A final extension step at 72°C was performed for 10 min. Amplification products were electrophoresed on 1.5% agarose gels and purified with the QIAEX II gel extraction kit (Qiagen, Inc., Mississauga, Ontario, Canada). Purified DNA fragments were sequenced by the chain termination method using the ABI 3730 Automated DNA Sequencer and analyzed using Sequence Navigator Software (both from PE-Applied Biosystems, Mississauga, Ontario, Canada).

Sequencing of genomic DNA from the affected proband demonstrated no mutations in the coding regions of the LPL gene (data not shown), but did reveal 2 novel missense mutations in the ALMS1 gene – a G→A transversion at nucleotide 1381 of exon 6 and a C→T transversion at nucleotide 11755 in exon 17. These mutations resulted in the replacement of valine by isoleucine at codon 424 (V424I, exon 6) and histidine by tyrosine at codon 3882 (H3882Y, exon 17). The proband's mother was heterozygous for the H3882Y mutation while the proband's younger sister was heterozygous for the V424I mutation, proving that the mutations were on opposite chromosomes in the proband. The proband's older sister was unaffected both clinically and molecularly. See Figure 1.

The missense mutation V424I was subsequently genotyped in 200 healthy unaffected Caucasian controls using the primer pair and PCR conditions for exon 6 amplification. The amplified product was digested with restriction enzyme AatII (New England Biolabs Inc., Ipswich, MA, USA). All healthy controls were homozygous for G at nucleotide 1381. Similarly, the missense mutation H3882Y was genotyped in 200 healthy unaffected Caucasian controls using the primer pair and PCR conditions for exon 17 amplification. The amplified product was digested using the restriction enzyme RsaI (New England Biolabs Inc., Ipswich, MA, USA). Among the 200 controls, only 1 control was found to be heterozygous at nucleotide 11755 C/T, giving an allele frequency of 0.9975 for the C allele and 0.0025 for the T allele at this position, based on the Hardy-Weinberg equation. This suggests that the second heterozygote mutation, 11755 C→T, is present in the general population, albeit at a very low frequency.

Prior to the recent discovery of ALMS1 mutations causative for AS, the diagnosis of AS was made solely based on phenotype. However, AS exhibits a great degree of phenotypic variability, even within families, thereby creating difficulties for a universal definition of AS 1718. Recently, Marshall et al defined AS using age-specific criteria 38. See Table 3.

Sensorineural hearing loss and congenital retinal dystrophy are two cardinal features of AS. In fact, pendular or searching nystagmus and photodysphoria are often evident before the age of 1 yr. There is initial loss of cone function followed by rod disintegration, ultimately leading to early blindness. By the age of 16, ~90% of affected individuals are blind 38. In addition, development of subcapsular cataracts may contribute to vision loss 36. Exudative retinopathy has also been reported in AS 12. Meanwhile, ~80 % of affected individuals will develop bilateral sensorineural hearing loss 3. This usually occurs at a later age in childhood and is characterized by the initial loss of high frequency sounds. Progressive deterioration in hearing occurs, and is sometimes accompanied by conductive hearing loss due to chronic otitis media or glue ear 3. These early changes in neurosensory capabilities have tremendous impact not only on the social development of the child but also on his/her adaptation to the external environment.

Although delay of cognitive development is not a common feature of AS, delay in developmental milestones is seen in ~45% of AS-affected children. Other neurologic manifestations may include absence seizures and general sleep disturbances 3. The frequency of mood and psychiatric disorders in AS-affected individuals has not been determined.

Individuals with AS often have distinctive facial features, such as round face, deep-set eyes, thick ears, dental anomalies, hyperostosis frontalis interna, and premature frontal balding. Their toes and fingers are typically short and stubby with no polydactyly or syndactyly while their feet are typically noted to be wide and thick 38.

Childhood obesity is present in over 95% of individuals with AS 3. However, both waist circumference and body fat percentage (as measured using dual-energy X-ray absorptiometry) negatively correlated with age, independent of BMI, indicating the possible recruitment of more metabolically active fat stores 2. The presence of hyperphagia has been controversial 34. Although childhood is often accompanied by rapid growth with height above the 50^th percentile before puberty, most individuals demonstrate a decreased adult height 23. This height discrepancy is supported by evidence for advancement of the bone age by 1–3 years prior to puberty as described by Marshall et al 3. As well, abnormalities of the insulin growth factor (IGF) system of affected patients have been demonstrated 13. Yet, the exact reasons for short stature remain to be determined.

Similar to the features of the metabolic syndrome seen in the general population, patients with AS also demonstrate hypertriglyceridemia and type 2 diabetes. Type 2 diabetes is diagnosed in over 80% of individuals above the age of 16 while insulin resistance and hyperinsulinemia have been demonstrated in individuals as young as 1 year old, even prior to the onset of obesity 338. Meanwhile, hypertriglyceridemia is seen in ~50% of affected individuals, with acute pancreatitis secondary to hypertriglyceridemia occurring in ~5% 3. Other endocrinologic manifestations of AS include hypothyroidism, hypogonadism (particularly among men), alterations in the onset of puberty, ovarian cysts and hirsutism (among females), and short stature with abnormalities in the IGF-growth hormone system 313.

Dilated cardiomyopathy (DCM), affecting ~60% of individuals, can occur at any age, but most typically during infancy. Although DCM is the most common underlying cause of death in the infantile period, survival with infantile-onset of DCM tends to be better than that for adult-onset DCM. Marshall et al showed that while one-third of adult-onset DCM patients died, ~74% of infantile-onset DCM patients survived 3. However, children and adults who have survived infantile-onset DCM are still susceptible to sudden recurrences 38. In addition to cardiac problems, AS-affected patients can have a variety of respiratory problems, including chronic asthma, sinusitis/bronchitis, alveolar hypoventilation and recurrent pneumonia 3.

Hepatic involvement in AS was first described in 1991 39. Approximately 80% of patients affected with AS may have hepatic involvement, ranging from mild elevation in liver transaminases to hepatic steatosis to overt cirrhosis with portal hypertension 33031. Other gastrointestinal effects include upper gastrointestinal pain, chronic diarrhea, constipation, and gastroesophageal reflux 3.

Renal insufficiency occurs in ~50% of AS-affected individuals. Whether hypertension is a consequence of renal insufficiency or contributes to renal insufficiency is uncertain, but it is present in ~30% of individuals 3. Urologic dysfunction is common in both men and women and can be manifest as urge incontinence, poor flow, urinary retention, or difficulty initiating voiding 3. Urologic anatomical abnormalities can also occur in AS, including calyceal deformities, narrowed ureteropelvic angles, dilated ureters, and misalignment of the kidneys 6.

It remains important to distinguish AS from other disorders characterized by childhood obesity and retinal dystrophy, such as the Laurence-Moon and Bardet-Biedl syndromes. Normal mentation and lack of poly/syndactyly help to distinguish AS from Bardet-Biedl, while deafness and the absence of spastic paraparesis help to differentiate AS from Laurence-Moon. Refsum disease is a rare disorder characterized by hearing loss, visual loss, and hepatic involvement, but also includes several features not associated with AS 40. See Table 4 for clinical features of these disorders.

AS is an autosomal-recessive disease that demonstrates intrafamilial and interfamilial variation. To date, a total of 81 disease-causing ALMS1 mutations have been reported, including the 2 reported from our group 11. A schematic representation of the normal ALMS1 gene and the ALMS1 mutations causative of AS are shown in Figures 2, 3, 4.

Affected individuals of most kindreds demonstrate compound heterozygosity with the majority of ALMS1 mutations reported being nonsense. Approximately 40% of all known ALMS1 mutations occur in exon 16 while other ALMS1 mutational hotspots include exon 10 (23%) and exon 8 (21%) 11. Mutations in exon 16 have correlated with a more severe disease phenotype, manifest as early onset of retinal degeneration before the age of 1 year (p = 0.02), presence of urologic dysfunction (p = 0.02), occurrence of dilated cardiomyopathy (p = 0.03), and development of diabetes (p = 0.03). Meanwhile, the severity of renal disease in AS has correlated with mutations in exon 8 11.

Among 250 AS-affected individuals examined, 15 synonymous single nucleotide polymorphisms (SNPs), 51 nonsynonymous SNPs, and a deletion of 3 nucleotides in exon 8 have also been described. Using prediction modeling, Marshall et al have determined that 8 of these variants are disease-causing 11. The most common ALMS1 mutation is c.10775delC, which is observed in 50% of reported English kindreds. Similarly, haplotype sharing has been demonstrated amongst members of Turkish kindreds, indicating the possibility of separate founder effects in both the English and the Turkish kindreds 11.

Our results indicate that ALMS1 mutations of V424I and H3882Y are the molecular basis for the Alstrom phenotype in the proband presented here. The H3882Y mutation was present in only 1 of 200 normal healthy controls while the V424I mutation was absent in all 200 controls. These two disease-causing mutations have not been reported before and represent the first mutation reported for exon 6 and the second reported for exon 17 11. Interestingly, the predicted frequency of homozygotes for the H3882Y mutation would be 1 in 160 000, signifying that, if homozygotes are affected, AS may indeed be more prevalent than previously thought.

To date, with the 2 missense mutations of our kindred, there are a total of 10 missense AS-causing mutations. Marshall et al showed that the majority of kindreds affected by AS, as in our kindred, demonstrate compound heterozygote mutations 11. The V424I mutation localizes to the N-terminus of the ALMS1 protein while the H3882Y mutation localizes to the C-terminus of the protein 7. Since even fragments of the N-terminus have been shown to be able to normalize the abnormal ciliary structure of ALMS1 knockout mice, it would be expected that isolated heterozygous mutations still including the relatively intact N-terminus would not necessarily result in significant clinical findings, as evidenced in our proband's mother and younger sister 10. The functional importance of the C-terminus remains to be determined. Importantly, no correlations could be drawn between the presence of a single ALMS1 mutation, as seen in the mother and younger sister, and abnormalities in any of the lipid parameters examined.

Hypertriglyceridemia was evident in our proband and is a common finding among individuals affected with AS. Our proband had no documented episodes of pancreatitis, but was at high risk for pancreatitis given her significant hypertriglyceridemia. There can be multiple factors contributing to hypertriglyceridemia among AS-affected patients, including insulin resistance or diabetes, liver dysfunction, renal dysfunction, dietary factors, and alcohol intake. However, examination of a group of AS-affected individuals demonstrated no significant correlations between hepatic or renal function, BMI, or degree of glucose intolerance and high TG levels. Instead, there was indeed a major correlation between hyperinsulinemia and hypertriglyceridemia in that study 5. Our proband demonstrated significant hypertriglyceridemia, accompanied by insulin resistance (type 2 diabetes), elevated BMI, and renal dysfunction. A multifaceted treatment approach resulted in improvements in TG and HDL-C to 3.30 mmol/L and 1.41 mmol/L, respectively, demonstrating that it is possible to effectively treat lipid abnormalities among AS-affected individuals.

The correlation of mutations in exons 6 or 17 to phenotype remains to be determined. Interestingly, our proband did not have sensorineural deafness, which typically occurs in ~84% of affected individuals 11. No genotype-phenotype correlations have been made for sensorineural deafness 11. However, it is possible that mutations in exon 6 or 17 may not be associated with sensorineural deafness although further kindreds with these mutations are required to substantiate this possibility. Yet, the mutations in this proband seem to have resulted in an "intermediate-severity" phenotype as judged by an early onset of retinal degeneration, severe renal dysfunction, and significant elevation of TG levels, but a later development of diabetes and a lack of dilated cardiomyopathy.