Alkaptonuria is a human autosomal recessive disorder caused by mutation of the HAO gene that encodes the enzyme homogentisic acid oxidase. A map of the HAO gene region reveals four BamHI restriction sites (B1 to B4) in the wild-type allele and three BamHI restriction sites in the mutant allele. BamHI utilizes the restriction sequence 5′-GGATCC-3′. The BamHI restriction sequence identified as B3 is altered to 5′-GGAACC-3′ in the mutant allele. The mutation results in a Ser-to-Thr missense mutation. Restriction maps of the two alleles are shown below, and the binding sites of two molecular probes (probe A and probe B) are identified.

DNA samples taken from a mother (M), father (F), and two children (C1 and C2) are analyzed by Southern blotting of BamHI-digested DNA. The gel electrophoresis results are illustrated.

Using A to represent the wild-type allele and a for the mutant allele, identify the genotype of each family member. Identify any family member who is alkaptonuric.

Verified step by step guidance

1. Step 1: Understand the genetic basis of alkaptonuria. It is an autosomal recessive disorder caused by mutations in the HAO gene. This means that individuals with two copies of the mutant allele (aa) will express the disorder, while individuals with one wild-type allele (A) and one mutant allele (Aa) are carriers and do not express the disorder.
2. Step 2: Analyze the restriction maps provided. The wild-type allele (A) has four BamHI restriction sites, while the mutant allele (a) has three BamHI restriction sites due to the alteration of the B3 site from 5′-GGATCC-3′ to 5′-GGAACC-3′. This change affects the digestion pattern observed in Southern blotting.
3. Step 3: Examine the Southern blot results for each family member (M, F, C1, and C2). The banding patterns on the gel correspond to the DNA fragments generated by BamHI digestion. Compare these patterns to the expected digestion patterns for the wild-type allele (A) and mutant allele (a).
4. Step 4: Assign genotypes based on the banding patterns. For example, individuals with bands corresponding to both wild-type and mutant alleles are heterozygous (Aa), while individuals with bands corresponding only to the mutant allele are homozygous recessive (aa) and are affected by alkaptonuria.
5. Step 5: Identify any family member who is alkaptonuric. Homozygous recessive individuals (aa) will express the disorder. Use the genotype assignments from Step 4 to determine which family member(s) are affected.

Key Concepts

Autosomal Recessive Inheritance

Autosomal recessive inheritance refers to a pattern where two copies of a mutated gene (one from each parent) are necessary for an individual to express a trait or disorder. In the case of alkaptonuria, individuals must inherit two copies of the mutant HAO gene to exhibit symptoms. Carriers, who possess one normal and one mutant allele, do not show symptoms but can pass the mutant allele to their offspring.

Restriction Enzymes and Restriction Mapping

Restriction enzymes, like BamHI, cut DNA at specific sequences, allowing researchers to create restriction maps that illustrate the locations of these cut sites. In this scenario, the presence of different numbers of BamHI sites in the wild-type and mutant alleles helps identify genetic differences. Analyzing these patterns through techniques like Southern blotting can reveal the genotypes of individuals based on the presence or absence of specific bands in the gel.

Missense Mutation

A missense mutation occurs when a single nucleotide change results in the substitution of one amino acid for another in a protein. In the context of alkaptonuria, the Ser-to-Thr missense mutation in the HAO gene alters the enzyme's function, leading to the accumulation of homogentisic acid. Understanding this mutation is crucial for determining the biochemical basis of the disorder and its inheritance pattern.